Adenovirus purification on glycerol caesium step gradients

Layer:

2ml 0.418 g/ml CsCl

3ml 0.32 g/ml CsCl

2ml 40% glycerol all above in TE

Layer freeze thawed Acton extract on top of step gradient and spin in the SW41 rotor for 90 minutes at 25,000 rpm at 40C.

COUPLING OF ANTIBODY TO PROTEIN A SEPHAROSE USING DIMETHYLPIMELIMIDATE

Reagents:

1. 200mM borate buffer + 3M NaCl, pH 9.0

2. 20mM dimethylpimelimidate in 200mM borate buffer + 3M NaCl, pH 9.0

3. 200mM ethanolamine, pH8 (1:80 dilution of stock ethanolamine)

4. Phosphate buffered saline (PBS)

5. PBS + 0.1% azide

6. 200mM glycine, pH 2.5

7. Protein A sepharose beads

Method:

1. Swell 250mg Protein A sepharose beads in 2ml PBS + 0.1% azide, at least one hour before use. (Yields a bed volume of 1ml beads)

2. Mix Protein A sepharose beads with antiserum for 2 hours at room temperature (using a rotator).

3. Centrifuge Protein A sepharose beads at 2500 rpm for 5 minutes.

4. Wash Protein A sepharose beads 2x in 200mM borate buffer + 3M NaCl.

5. Resuspend Protein A sepharose beads in 20mM dimethylpimelimidate.

6. Rotate Protein A sepharose beads for 30 minutes at room temperature.

7. Centrifuge Protein A sepharose beads at 2500 rpm for 5 minutes.

8. Wash Protein A sepharose beads 2x in 200mM borate buffer + 3M NaCl.

9. Wash Protein A sepharose beads 1x in 20mM ethanolamine.

10. Resuspend Protein A sepharose beads in 20mM ethanolamine and rotate for 2 hours at room temperature.

11. Centrifuge Protein A sepharose beads at 2500 rpm for 5 minutes.

12. Wash Protein A sepharose beads 2x in PBS.

13. Wash Protein A sepharose beads 2x in 200mM glycine.

14. Wash Protein A sepharose beads 2x in PBS.

15. Store Protein A sepharose beads in PBS + 0.1% azide. (1 volume PBS to 1 volume beads)

Nuclear and cytoplasmic cell extractions

Author: Kate (from Fernando)

Project: NF-kB

Date: 9-9-93

Reference

Method (Mg63 cells)

Day 1

1 Serum starve subconfluent flat of Mg63 cells (for activation studies ~2 x 75 cm2 flats needed for each time point) by replacing GMEM:5% FCS with GMEM alone. Incubate 24 h, 37 oC, 5% CO2.

Day 2

2 Dilute Tumour Necrosis Factor-a (TNF, 100 ng.µl-1 stock solution in H2O, -20 oC) in warmed GMEM to give 10 ng.ml-1 (1 µl TNF + 10 ml GMEM for each 75 cm2 flat).

3 For zero time point, aspirate off old GMEM and replace with 1 ml fresh GMEM.
For other time points, replace GMEM with GMEM/TNF.
Begin timing.

4 At suitable time points(eg 10 min, 45 min, 2 h), stop activation by replacing GMEM/TNF with ice-cold PBS:25 µM TPCK (made fresh, 125 µl of 25 µM TPCK stock in 50 ml PBS). Wash cells 1x in same buffer.

5 Scrape cells into minimum volume of PBS:25 µM TPCK using rubber cell scraper.

6 Harvest cells by centrifugation (6000 rpm, 2 min, 4 oC, eppendorf centrifuge 5402, Lab 28).

7 Wash cells 2x by resuspending pellet in 500 µl PBS:25 µM TPCK:1 mM PMSF (made fresh) and centrifugation, as before.

8 Gently resuspend pellet in 50 µl Buffer A:25 µM TPCK:1 mM PMSF (see sheet).
Leave on ice 2 min.

10 Centrifuge as before (6000 rpm, 2 min, 4 oC, eppendorf centrifuge 5402, Lab 28) and remove supernatant (cytoplasmic fraction) to fresh tube.

11 Re-extract pellet in 50 µl Buffer A:25 µM TPCK:1 mM PMSF, centrifuge as before and pool the two cytoplasmic extracts. Remove last traces of Buffer A using fine pipette tip.

12 Wash pellet in high salt buffer by gently mixing the pellet with 100 µl Buffer B:25 µM TPCK:1 mM PMSF. Do not resuspend vigorously.13 Remove Buffer B by brief centrifugation (30 sec, 6000 rpm, 4 oC) and aspiration using fine pipette tip.

14 Resuspend pellet in 15 µl of Buffer C:25 µM TPCK:1 mM PMSF.
Leave on ice, 20 min, tapping at intervals.

15 Centrifuge at high speed (5-15 min, 4 oC, 14,000 rpm, eppendorf centrifuge 5402, Lab 28).

16 Collect supernatant (nuclear extract).

17 Estimate protein concentration in nuclear and cytoplasmic extracts using Bradford's reagent. Samples may be stored at this stage (-70 oC).

18 For Western blot examination using @IkB antisera, 80 µg nuclear extract and 40 µg cytoplasmic extract suitable (see SDS-PAGE, Western blotting and ECL protocols).

Notes Buffers containing protease inhibitors are made immediately before use since 1/2 life PMSF in aqueous solution only minutes.

Solutions PMSF (Sigma):

Stock solution = 100 mM in propan-2-ol, 20 oC
(Mr= 174.2 g, 100 mM= 174.2 mg in 10 ml propan-2-ol)
Working conc. = 1 mM, 50 µl stock in 5 ml PBS

TPCK (Sigma):

Stock solution = 0.5 M TPCK in DMSO, -20 oC
(Mr= 351.9 g, 0.5 M= 175.95 mg in 1 ml DMSO)
Working stock = 10 mM TPCK in DMSO, 1/50 dilution, 10 µl in 500 µl
Working conc. = 25 µM, 12.5 µl working stock in 5 ml PBS.

 

SDS-PAGE

Author Kate (from Ron)
Project Nf-kB
Date 21-9-93
Reference Bio-Rad Mini-protean II dual slab gel instruction manual
Method For Bio-Rad mini gels:
1 Using gel casting apparatus, assemble gel plates according to manufacturers instructions. To prevent gel leakage, ensure that the two glass plates and spacers are flush at the base.
2 Prepare suitable %age gel by combining the following components in a small flask, adding polymerisation catalysts (TEMED and AMPS) last:

		Volume (ml)
	15%	10%			8%
	(x1)	(x1)	(x2)	(x1)
GB	1.2	1.2	2.4	1.2
30% acrylamide stock (29:1, Acryl:Bis Integra Biosystems)	2.6	1.7	3.4	1.3
H2O	1.2	1.7	3.4	1.3
APS	0.03	0.03	0.06	0.03
TEMED (Sigma)	0.003	0.003	0.006	0.003
(Total)	(5)	(5)	(5)	(5)

3 Pour mixture between gel plates, leaving 2 cm space at the top. Overlay with 1 ml dH2O and allow gel to set (30 min-1 h).
4 Prepare stacking gel:
Stacking gel:

	Volume (ml)
	(x1)	(x2)
SGB	0.4	0.8
30% acrylamide stock	0.6	1.2
H2O	1.4	2.8
APS	0.01	0.02
TEMED	0.001	0.002

5 Once separating gel set, pour off overlay and add stacking gel. Insert comb, taking care not to trap bubbles beneath teeth. Allow to set, 30 min.
6 Remove comb, rinse out wells with dH2O. Place clamp assembly into inner cooling core, ensuring small glass plate is in contact with notch in gasket.
Notes
Solutions
SGB 100 ml GB 100 ml
Tris 5.1 g Tris 18.5 g SDS 0.4 g SDS 0.4 g
pH to 6.7 with HCl pH to 8.9 with HCl Make up to 100 ml with dH2O make up to 100 ml with dH2O

10X TGS 1 l Boiling mix
Tris 63.2 g SGB 1 ml Glycine 40 g 25% SDS 0.8 ml SDS 10 g β-mercaptoethanol 0.5 ml glycerol 1 ml Bromophenol blue 0.05%
Make up to 100 ml with H2O
Stain
Coomassie brilliant blue (Sigma) 4 g Methanol 1 l (add 1st) Glacial acetic acid 200 ml H2O 1 l

Preparation of nuclear and cytoplasmic extracts

(gives nuclear extract essentially free of cytoplasmic contamination)

Wash cells twice in ice cold PBS and collect by centrifugation in a microcentrifuge tube. To the cell pellet (approx 5 x 106 cells) add 200 µl buffer A + protease inhibitors and vortex. Spin out nuclei on refrigerated microfuge 5k/2min. Remove SN and keep as the cytoplasmic extract. Wash the nuclear pellet by resuspending in 500 µl buffer B + protease inhibitors. Discard SN and take up nuclear pellet in 50 µl buffer C + protease inhibitors. Incubate on ice for 30 min. with frequent agitation. Spin 15 min, full speed, in refrigerated microfuge. Carefully remove the SN and store as the nuclear extract.

Buffer A

50 mM NaCl, 10 mM HEPES pH 8.0, 500 mM sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.2 % TX-100.

Buffer B

50 mM NaCl, 10 mM HEPES pH 8.0, 25 % glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine.

Buffer C

350 mM NaCl, 10 mM HEPES pH 8.0, 25 % glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine.

Add protease inhibitors and mercaptoethanol to 7 mM to all buffers just before use.

Protease inhibitors

1 mM PMSF, 2 mM benzamidine, 1 µg/µl leupeptin, 1µg/ml pepstatin, 0.2 IU/ml aprotinin. Alternatively you can buy tablets from Boehringer Mannheim that contain a cocktail of inhibitors - just add to your buffer (they work pretty well).

Northern analysis of RNA

 

RNA preparation.

Use one of the commercially available kits that employ GuHCl to get complete lysis of cells and inactivation of RNases.

Alternatively make cytoplasmic RNA as follows

(for one 90 mm plate of cells)

1. wash monolayer x2 with STE

2. Scrape into 2 ml of STE, remove to a sterile tube, wash plate with a further 1ml and combine with 2 ml in tube.

3. Spin, 1K/5min/40C and discard SN

4. Flick tube to disperse cells, add 200 µl RNA lysis buffer, then 20 µl 10% NP40

5. Vortex then spin 5K/5 min

6. Remove SN and transfer to Ep tube containing 200 µl STE + 2% SDS

7. Extract x3 with 1:1 phenol chloroform, then x1 chloroform.

8. Add NaOAc to 0.3 M and ethanol precipitate.

9. Repeat step 8 above.

10. Take up in 20 µl H2O (DEPC treated)

11. Determine OD260 (2µl -> 500 µl).

STE is 150 mM NaCl, 10 mM Tris HCl pH 7.5, 1 mM EDTA

RNA lysis buffer is 10 mM Tris HCl pH 7.8, 10 mM NaCl, 2 mM MgCl2

 

Formaldehyde gels

for 100 ml

Add 60 ml H2O to 1.5 g of agarose and microwave to dissolve

Add 20 ml 5x MOPS buffer and 20 ml formaldehyde (stock is 37% soln.)

Pour into gel mould, allow to set and place in gel rig with 1x MOPS bufer

 

RNA

5.5 µl RNA in H2O (DEPC treated) - run 10 - 30 µg total RNA

1 µl 5x MOPS buffer

3.5 µl formaldehyde (37% soln.)

10 µl formamide (deionized by mixing with MB1 resin)

Incubate at 600C for 5 min.

Add 2 µl formamide containing BPB as dye

Load onto gel and run at 25 mA for 60 min. then at 50 mA.

5X MOPS bufer is 100 mM Na MOPS (morpholino-propane sulphonate), 25 mM NaOAc, 5 mM EDTA, pH to 7 with HAc

At end of run wash in H2O (DEPC treated) for 30 min then 20x SSC for 30 min

Transfer RNA to nitrocellulose by blotting in 20x SSC

Bake at 800Cin a vacuum oven for 2 hr.

Synthesis of Nickel (Ni-NTA) Sepharose for purification of His-tagged proteins

Chemicals

Bromoacetic acid, Ne-benzyloxycarbonyl-L-lysine, palladium on charcoal, epibromohydrin and Sepharose CL-6B can be obtained from Sigma Aldrich.

Note hydrogenation apparatus is required for the deprotection step.

 

Method

1. Dissolve bromoacetic acid (41.7 g) in 150 ml 2 M NaOH and cool to 0 ºC.

2. Prepare a solution of Ne-benzyloxycarbonyl-L-lysine (42 g) in 2 M NaOH.

3. Add the protected amino acid solution to the bromoacetic solution slowly dropwise at 0 ºC with stirring.

4. After 2 hours remove the cooling and continue stirring the mixture overnight.

5. Heat the mixture to 50 ºC for 2 hours.

6. Add 450 ml 1 M HCl and cool the mixture in ice.

7. Filter off the crystals produced, dissolve them in a minimum volume of 1 M NaOH and reprecipitate with 1 M HCl.

8. Filter off the crystals and allow to dry.

9. Dissolve a 7.9 g portion of the above product in 49 ml 1M NaOH , add a small portion of 5%Pd on charcoal and hydrogenate at room temperature and pressure.

10. When the hydrogenation has gone to completion filter off the catalyst and concentrate the filtrate by rotary evaporation.

11. Wash a 100 ml volume of Sepharose CL-6B twice on a glass suction filter with 500 ml water.

12. To activate the Sepharose heat it to 30 ºC for 4 hours with 16 ml 4 M NaOH and 8.22 ml epibromohydrin.

13. Filter off the Sepharose and wash to neutrality with water.

14. Dissolve 6 g of the hydrogenated reaction product in 50 ml water and add to the activated Sepharose together with 10.6 g solid sodium carbonate. Stir the reaction mixture slowly at 60 ºC overnight.

15. Filter off the Sepharose and wash with water.

16. To charge the resin with nickel wash through slowly with 100 ml aqueous NiSO4, 200 ml water, 200 ml 0.2 M acetic acid containing 0.2 M sodium chloride and 0.1% Tween-20 and finally 200 ml water.

The resin should bind up to 10 mg/ml of tagged protein.

Ref. Hochuli E, Dobeli H and Schacher A, 1987, J Chromatography, 411, 177-184.

 

KATRINA’S METHOD FOR MONOCLONAL ANTIBODY PURIFICATION

1. Swell 100mg Protein A sepharose in 400µl PBS at least one hour before use.

2. Clarify monoclonal antibody producing supernatant by centrifugation at 4000rpm for 10 minutes.

3. To 50ml serum-free supernatant, add solid NaCl and glycine to a final concentration of 3M and 1.5M, respectively, and adjust the pH to 8.9.

4. Incubate Protein A sepharose with supernatant for 3 hours at ambient temperature on a rotator. Alternatively, circulate supernatant over the column overnight at 4oC, using a peristaltic pump.

5. Remove the supernatant (either by centrifugation or draining the column) and wash the Protein A beads with 100ml of binding buffer (3M NaCl, 1.5M glycine, pH 8.9).

6. Elute the column with 100mM citric acid, pH3 (adjusted with 4M NaOH).

7. Collect 200µl fractions onto 50µl to 100µl Tris/HCl, pH 8, in eppendorf tubes so the pH is restored to neutral.

8. Load 5µl of each fraction onto an SDS-Page gel (+10µl of starting supernatant).

9. Dialyse positive fractions overnight against PBS with 3 changes of PBS.

Microsequencing gel purified proteins

NB. Extreme care and cleanliness is required or you will end up sequencing keratin from skin - wear gloves at all times

Run gel as for sequencing ie freshly made up acrylamide, polymerised overnight,include scavengers and prerun.

Place in a new, sterile, square petri dish and stain in CBB (5 min)

Destain (until bands are clear)

Using a new scalpel excise CBB stained band. As a control cut equivalent pieces of the gel that do not have stained protein.

Place each gel band in a sterile screw cap microfuge tube. Add 1 ml water (ultrapure). Agitate and change water a few times over a period of 60 min

Remove all traces of water and freeze dry overnight.

To the gel slice add 200-300 mL of 50 mM TrisHCl pH 8.0, 0.1% reduced Triton X100 containing 1 mg trypsin (sequencing grade). Digest overnight at 370C.

Remove SN and apply to microbore HPLC.

Compare with blank and sequence unique peaks.

MALDO-TOF mass spec analysis

Proteins and peptides should ideally be presented to the service at a concentration of at least 10 pmol/microlitre in 0.1 % TFA or water. (10 pmol/microlitre is equivalent to 0.2 mg/ml for a 20 kDa protein, 0.4 mg/ml for a 40 kDa protein and so on.) The buffer and its components, especially salt, detergents and glycerol, are detrimental to signal quality. However, we have a simple dialysis system which requires only 5 microlitres of sample and can be performed directly before sample loading thus minimising any solubility problems. We will perform this dialysis on any samples we consider require it or for which it may improve the result, at our own discretion and charge appropriately. Samples of less than 10 pmol/microlitre can be analysed after multiple application on the spot or concentration on a ZipTip (small reverse phase column), but recovery cannot be guaranteed from the latter. 5 - 10 microlitres of sample is required, to allow for dialysis etc. The basic charge is for external calibration, based on one sample and one set of calibrants. Reduced charges will be made for multiple samples of similar mass which can be calibrated using the same external calibrants. Additional charges will be made for internal calibration or running with different matrices. These will be performed at our discretion in order to help answer the question posed, e.g. purity levels, modifications, etc.

The major ion observed is the M+H ion. In addition M+Na, M+K and M+matrix peaks may be seen.

Proteins of up to about 150 kDa can be accommodated. However, with increasing size the resolution deteriorates such that, although the mass accuracy is still acceptable, two species of close molecular weight may not be distinguishable. Although protein mixtures can be analysed it should be noted that smaller species have a suppression effect on larger species, such that the larger protein in a mixture may not give a signal. In no way do relative peak intensities bear any relationship to relative concentration.

Peptides of mass <6 kDa can be determined to better accuracy than larger molecules as they can be measured in reflectron mode. Greatest accuracy can be obtained by internal calibration. However, for the reasons given above, this is not always feasible and external calibration will then be used.

We can identify proteins (that are in the SWISS-PROT or TREMBL databases) from bands or spots on SDS-PAGE gels by in gel digestion with trypsin, peptide extraction, analysis of the peptide mix by mass spectrometry and database searching.

Sequence information can be obtained from peptides by post source decay (PSD) or C-terminal protease ladder sequencing. Results are very peptide dependent.

Oligonucleotides (up to 30 mers) can be analysed. They should be presented at 5 pmol/microlitre in water.

Luciferase assays

NB Do not wear gloves and do not touch the walls of the cuvettes.

(quantities for 1 x 50 mm plate of cells)

1. Wash cells x 2 with 4ml PBS (ice cold) and drain.

2. Add 200µl Lysis buffer (ice cold) and leave on ice for 5min.

25mM Tris phosphate pH 7.8

8mM MgCl2

1mM DTT

1% Triton X100

15% Glycerol

3. Scrape cells from plate and transfer to a 1.5ml Eppendorf tube.

4. Determine protein concentration by Bradford assay using BSA as standard

5. Add 100µl of cell extract, or lysis buffer as blank, to a cuvette and place in luminometer.

6. Inject 100µl of luciferase buffer

1mM ATP

0.25 mM luciferin

1% BSA

made up in lysis buffer.

7. Read relative light units and correct for blank and protein concentration.

Immunoprecipitation

(This procedure is not suitable for precipitation of multiprotein complexes where protein-protein interactions have to be preserved)

Materials

Lysis buffer

25mM Tris HCl pH 8.8

50mM NaCl

1% SDS (use when antigen has to be completely denatured for antibody recognition)

RIPA buffer

25mM Tris HCl pH 8.8

50mM NaCl

0.5% NP40

0.5% Deoxycholate

0.1% SDS

Before use add BSA to 0.1%, protease inhibitors and phosphatase inhibitors.

PAS

50% protein A slurry 25mM Tris HCl pH 8.1, 144mM NaCl

Procedure (stringent procedure for rare proteins eg. transcription factors)

10cm dish of cells

incubate for 45min in Met free medium, wash

Label with 1mCi 35S Met for 2hr in 2ml Met free medium

Remove medium, wash PBS

Add 1ml disruption buffer (either RIPA or lysis buffer)

scrape, (boil for 2min if using lysis buffer)

shear 6x in a 25 gauge needle

Microfuge for 5 min.

(if using lysis buffer dilute x10 with RIPA buffer without SDS)

Preclear by adding 4µl normal rabbit serum and 40µl PAS

Rotate for 30 min, spin 30s.

Remove SN which can be split up at this stage

to about 500µl add 2µl immune serum and 20µl PAS (first incubate this slurry in cold lysate for 60min and wash x3 in RIPA buffer + BSA.

Rotate 2hr at 40C

Spin, transfer to 15ml tube (optional, use for very low abundance proteins) and wash x3 with 10ml RIPA + BSA

transfer back to microfuge tube

spin 30s, aspirate to dryness with very fine needle

Add gel loading buffer, boil, spin and load SN onto gel.

1) Immunofluorescence — Catherine Dargemont

For immunofluorescence, the protocol I use is the following

1. Wash in PBS++( + 1 mM MgCl2, 0.5 mM CaCl2)

2; 3% paraforfaldehyde, 10 min

3. 3 washes in PBS

4. 2x 10 min PBS/0.1M glycine

5; 1 wash PBS

6. 0.2 % TX100, 10 min

7. 2 washes in PBS+ 1 wash in PBS/BSA 0.2%

8. 1 antibody diluted in PBS/BSA

9. 3 washes in PBS/BSA

10. anti sheep antibody-FITC diluted in PBS/BSA

11. 2 washes in PBS/BSA+ 1 wash in PBS

12. Mounting

 

2) Immunofluorescence with non-suspension cells (Mg63)

Author Kate (from Liz)

Project NFkB

Date 2-9-93

Reference

Method Day 1 (preparation)

1 Using immunofluorescence forceps (Harwell), remove individual glass cover slips from storage in EtOH, flame briefly and distribute in a 24 well-plate

2 Trypsin-treat confluent 75 cm2 flat of Mg63 cells (see tissue culture protocol) and resuspend cells in 10 ml GMEM:5% FCS

3 Transfer 2 ml into new flat and make up volume to 50 ml with GMEM:5% FCS (remaining 8 ml can be distributed between 4 flats [2 ml in each made up to 25 ml] and used in cell extraction protocol, or discarded).

4 Place 1 ml of resuspended cells into each well of the 24 well plate, the remaining 26 ml can be used as a stock of Mg63 cells.

5 Grow 48 h in 5% CO2 incubator (37 oC).

Day 3 (serum starvation)

6 If cells subconfluent (~60-80%), carefully aspirate off media, wash x1 with GMEM alone (37 oC) and replace with a further 1 ml GMEM. Grow under serum-free conditions ('serum starvation'), 24 h (5% CO2, 37 oC)

Day 4 (activation)

7 Dilute Tumour Necrosis Factor (TNF) in warmed GMEM to give 5-10 ng.ml-1 ( 1 µl + 10 ml GMEM). (TNF stored as a stock solution [-20 oC], 100 ng.µl-1. Manufacturer states TNF active between 0.05-20 ng.ml-1).

8 For zero time point, aspirate off old GMEM and replace with 1 ml fresh GMEM.
For other time points, replace with GMEM containing TNF.
Begin timing.

9 At suitable time points (eg 0, 10 min, 45 min, 2 h), remove cover slips, plunge into a beaker containing ice-cold PBS, and place into 24-well plate containing ice-cold fix.

10 Replace fix with storage buffer after 10 min. Cover slips can be stored for some time (1-2 weeks?) at 4 oC in storage buffer.

Immunofluorescence

11 Aspirate off storage buffer and wash 2 x PBS.

12 Remove PBS and add 1 ml permeabilisation buffer. Leave 5 min 20 oC.

13 Wash 3x dilution buffer.

14 Prepare suitable dilution of 1o antibody in dilution buffer.
For identification of IkB in Mg63 cells, an antibody concentration of 2 µg.ml-1 (affinity purified polyclonal @IkB) gave detectable fluorescence with low levels of background fluorescence from control antisera (affinity-purified rabbit @IkB pre-bleed).

15 Spot 8 µl antisera onto clean microscope slides.

16 Carefully remove XS liquid from fixed and washed cover slips and place, cells downwards, onto spots of 1o antisera.
Incubate 1 h, 20 oC in damp box.

17 Transfer cover slips to 24 -well plate containing Dilution buffer. Leave 5 min, 20 oC.
Replace with 1 ml penetration buffer, 5 min 20 oC.
Wash 3x dilution buffer.

18 Dilute 2o antibody in Dilution buffer.
(For detection of above 1o antisera, 1/100 dilution of DTAF@rabbit [Jackson Labs] suitable).
Spot 8 µl onto microscope slides, taking care to minimise exposure of antisera to light.

19 As before, place cover slips onto spots of 2o antisera.
Incubate 1 h, 20oC in damp box (dark).

20 Wash cover slips in 1x Dilution buffer
1x Permeabilisation buffer
3x Dilution buffer
2x PBS

21 Mount cells onto microscope slides containing 1 µl spots of 'Citimount' and seal with nail varnish. Store in dark dry box until nail varnish dry.
Examine with blue fluorescence.

Notes Washes may be performed by dipping coverslips (10x) into beakers containing suitable wash buffers.
Since Mg63 cells appear to dislodge by this method, cells are washed by careful addition of 1 ml buffer, incubated 5 min (20 oC) and buffer remove by careful aspiration. Although more time-consuming, the washing method is less harsh.

Solutions Fix

PBS 500 ml
5% formaldehyde
(v/v of a 40% solution) 25 ml
2% sucrose 10 g

Permeabilisation buffer

PBS 500 ml
0.5% NP40 2.5 ml
10% sucrose 50 g
1% Donkey serum (SAPU) 5 ml

Dilution buffer

PBS 500 ml
Donkey serum 5 ml

 

Hi-TRAP COLUMN

Buffers and reagents:

Coupling buffer: 33.6g Na HCO3 (final concentration 200mM) + 58.44g NaCl (final concentration 500mM) made up to 2 litres with H2O

Buffer A: 1.527g ethanolamine + 1.461g NaCl made up to a final volume of 50ml with H2O after adjusment of pH to 8.3 [final concentration of ethanolamine and NaCl are 500mM and 500mM, respectively]

Buffer B: 410mg acetate + 1.461g NaCl made up to a final volume of 50ml with H2O after adjustment of pH to 4.0 [final concentration of acetate and NaCl are 100mM and 500mM, respectively]
PBS
1mM HCl
10mM Tris, 500mM NaCl, pH 7.5
100mM Glycine, pH 2.25
1M Tris, pH 8.0
PBS, 500mM NaCl
PBS, 500mM NaCl, 0.1% azide

Preparation of Hi-Trap Column (1ml):

1. Apply 1 drop of 1mM HCl on top of the column to prevent air bubbles.
2. Wash the column six times with 1mM HCl.
3 Wash the column ten times with coupling buffer.
4. Pump on ligand (about 6mg of recombinant IkB, p65 or p50).
5. Recirculate to make sure all material has bound to the column.
6. Seal the column and let stand for 15-30 minutes.
7. Deactivate any excess groups that have not coupled to ligand and wash out the non-specifically bound ligand by the following procedure:

a. pass 6ml Buffer A through the column
b. pass 6ml Buffer B through the column
c. pass 6ml Buffer A through the column
d. let the column stand for 15-30 minutes
e. pass 6ml Buffer B through the column
f. pass 6ml Buffer A through the column
g. pass 6ml Buffer B through the column

8. Store column at 4oC in the presence of 0.1% azide until use.

 

Purification of Antibody

1. Wash Hi-trap column with 20ml coupling buffer.
2. Dilute 8ml serum to 80ml with PBS.
3. Centrifuge serum to remove any debris.
4. Load sample onto the column slowly and recycle flow through.
5. Wash column with 20 ml 10mM Tris/500mM NaCl, pH 7.5.
6. Keep wash.
7. Elute column with 100mM Glycine, pH 2.25.
8. Collect 500ml fractions into eppendorf tubes containing 50µl 1M Tris, pH 8.0.
9. Wash column with PBS/500mM NaCl (20 volumes).
10. Stores column at 4oC in PBS/500mM NaCl + 0.1% azide.
11. Perform protein assay on fractions to determine fractions containing antibody.
12. Pool fractions and dialyse against PBS overnight at 4oC.
13. Do protein assay on each set of pooled fractions.
14. Add protease-free BSA to a final concentration of 1mg.ml-1 to the protein containing fractions.
15. Aliquot and store antibody at -20oC.

Gradient mini gels

For 5-15% gradients
Gel mixes

Component	5%	15%
Gel buffer	2.4 ml	2.4 ml
30% acrylamide (29:1)	1.7 ml	5.2 ml
water	5.9 ml	1.4 ml
glycerol	1.0 ml	-

Solutions for layering

Final conc.	5% mix	15% mix
5%	2.0 ml	-
7.5%	1.34 ml	0.66 ml
10%	1.0 ml	1.0 ml
12.5%	0.66 ml	1.34 ml
15%	-	2.0 ml

Make up the 5, 7.5, 10, 12.5 and 15% solutions (2 ml) and place on ice. Once they have coolled add
0.5 µl TEMED
4.0 µl APS (25%)
Layer 0.7 ml of each solution into the gel using a 1ml syringe with the needle attached to a narrow piece of tubing that can reach to the bottom of the gel plates. Overlay with water and leave to polymerise. Add 5% stacking gel and proceed as normal.

 

DNA sequencing of GC rich regions

There are several ways to sequence through GC rich tracts, however not one method will generally work for all templates.

As a first attempt I would try using up to a maximum of 5% DMSO and 5% glycerol final conc in your sequencing reaction. The inclusion of glycerol helps to stabilise the enzyme. It is also important that fresh DMSO is used. Use a new bottle of dmso and aliquot into one shot aliquots, these should be stored at -20. As DMSO is a cosolvent it may also be necessary to lower the anneal temp of your primers by 2-3 deg.

If you are sequencing GC rich plasmids it may also be beneficial to linearise the plasmid first and use a linear template in your reaction.

Increasing the enzyme concentration may also help, if you are using a ready reaction kit this means doing a 2X reaction vol with twice the amount of the reaction components. If possible it is best to sequence using forward and reverse primers, as one primer may produce better results. If you use a 2X reaction vol this can also be combined with increasing the denaturation temp to 97-98 deg. However such high temps will denature the enzyme fairly rapidly. These higher temps should also not be used in combination with DMSO. In the worst case scenario if all the above fails you may need to redesign a new primer closer in toward the region of secondary structure.

Protein footprinting by the combined use of reversible and irreversible lysine modifications for protein-protein and protein-DNA interaction

[protocol for (p50)2+DNA, (p50)2+Ank/Ik Ba ]

Materials

protein: preferred buffer: in 20 mM sodium phosphate pH 8.3

if unstable in this buffer, try another one but always without chemicals with free amino group like Tris, or try to add some NaCl [i.e. p65 in PBS, NaCl, DTT, EDTA]

immunotagged at one end (C- or N-terminus): only for the protein of interest

DNA: in 20 mM sodium phosphate pH 8.3 (via ethanol precipitation if necessary)

reagents: Citraconic anhydride (Pierce): abbreviation CA

N-Hydroxysuccinimide acetate (Sigma): abbreviation NHSA, highly hygroscopic, stable for only a few weeks

Dioxan (Aldrich)

Endoproteinase Lys-C (Sigma or Promega)

 

Method

Protein concentrations in the reaction mixture: protein+DNA: 0.75 mg/ml

protein+protein: 0.80 mg/ml

for both types of experiments: same amount of protein of interest [i.e. 15 m g p50ctag]

Reaction buffer: 20 mM sodium phosphate pH 8.6

Incubation of proteins for formation of complexes as necessary [i.e. p50ctag+DNA: 10 min at room temperature, p50ctag+Ank: 15 min at 37° C]

Rapidly dilute CA to 0.5 mM by adding 4.5 m l of the reagent to 100 ml 20 mM sodium phosphate pH 8.6, under vigorous stirring at room temperature

Add the diluted reagent immediately to the reaction mixtures to give a CA/lysine molar ratio of 0, 0.15, 0.3; concentration of CA in reaction mixtures should be between 0.12 and 0.15 mM (adjust via volume of reaction mixture),

incubate the mixtures at room temperature for 30 min

Quench the reaction by adding an excess of Tris/HCl buffer pH 8.5 (about 80 to 90 mM or 500fold to CA), for 10 min at room temperature

Acetone precipitation with ice-cold (-20° C) acetone: add 2 vol. of acetone to samples to get 67% acetone, 30 min on ice, spin for 25 min at 14.000 rpm and 4° C, wash pellet with 1 vol. ice-cold acetone, spin for 8 min, air-dry at room temperature

Dissolve pellet in 1 vol. 7 M guanidinium chloride (buffered with 20 mM sodium phosphate to pH 8.4), at least 30 min at room temperature, vortex and spin down [1 vol. = 100 m l for 15 - 30 m g protein]

add 0.2 vol. 20mM sodium phosphate pH 8.2

Irreversible modification by adding 0.25 vol. of 2% NHSA/dioxan (127 mM, freshly prepared, used immediately), incubate at room temperature for 30 min

Terminate the reaction by adding 0.4 vol. 0.5 M Tris/HCl pH 8.5, 10 min at room temperature

Dialyse samples against 30% acetic acid in Microdialysis System (BRL) for 20 to 22 hours at room temperature

Use the spinning evaporator under reduced pressure to remove the acetic acid from the recovered samples

Dissolve pellet in 10 m l 8 M urea (freshly prepared, deionized with monobed mixed resin), at least 30 min at room temperature, vortex, spin down
add 30 m l 0.13 M Tris/HCl pH 8.5 (volumes regardless of amount of protein, final concentrations: 2 M urea, 0.1 M Tris/HCl)

Digest by adding endoproteinase Lys-C (Sigma or Promega): ratio to amount of protein the reaction was started with: 1:150 to 1:250

incubate at 37° C for 20 hours

Analysis by SDS-PAGE (12.5% or 15%) and Western Blot

for Western Blot: dilute 1 m l of digestion mix 25fold, apply 4 m l of this dilution on gel

NF-kB DNA binding assay

Binding buffer (Aliqot and store at -200C)

Component	Final conc.	conc. of stock	volume of stock
Hepes	25 mM	1.0 M	0.5 ml
EDTA	1 mM	0.5 M	0.04 ml
Spermidine	3.5 mM	0.5 M	0.14 ml
MgCl2	6 mM	1.0 M	0.12 ml
NaCl	100 mM	5.0 M	0.4 ml
NP40	0.15%	10%	0.3 ml
Glycerol	10%	100%	2.0 ml
H20	-	-	16.5 ml

Assay

Volume	Component	conc. of stock
18 µl	binding buffer	-
2 µl	DTT	100 mM, in MES pH6
2 µl	BSA	10 mg/ml in H2O
1 µl	non-specific DNA	0.5 µg/µl poly dAT 0.5 µg/µl poly dGC 50 ng/ul NFIII ds.oligo or other unrelated ds oligo
1-3 µl	protein extract	10 µg total
Incubate at 200C for 15 minutes
1 µl	32P labelled DNA	10,000 cpm, approx.

Incubate at 200C for a further 15 minutes
Add 4 µl GRA dyes (1.0 ml binding buffer, 1.0 ml glycerol, BPB to taste, store at -200C)
Load onto a 6% native polyacrylamide gel in 0.5x TBE

volume	component	conc. of stock
7.5 ml	acrylamide	44% acryl, 0.8% bis
2.5 ml	TBE,Tris-borate buffer	1 M (10x)
40 ml	H20	-
20 µl	TEMED	neat
200 µl	APS	25%

Run gel at about 200 V for 90 minutes using 0.5 x TBE as tank buffer.

Separate plates and press a single sheet of DE81 paper onto the gel surface. Using a piece of 3MM paper as backing place in drier and then expose to phosphorimager for a couple of hours or overnight with a film plus screen.

ELISA FOR p50 DETECTION

Reagents: Carbonate buffer, pH 9.5 (4.3g NaHCO3 and 2.15g Na2CO3)

Blocking buffer - 10% milk in PBS

Washing buffer - PBS + 0.01%Tween 20

Anti-mouse HRP (1:5000 in PBST)

TMBlue

PBS

p50 recombinant protein

 

Method:

1. Dilute p50 in carbonate buffer to a final concentration of 5µg.ml-1.

2. Aliquot 100µl into each well of a microtitre plate and incubate overnight at 4oC.

3. Wash the plate 3x in washing buffer.

4. Aliquot 200µl blocking buffer into all wells of the microtitre plate and incubate for 1 hour at 37oC.

5. Wash the plate 3x in washing buffer.

6. Aliquot 50µl into all wells except column 1

7. Aliquot 100µl primary antibody (or hybridoma supernatant) to wells of column 2.

8. Double dilute across the plate by removing 50µl from column 2 and mixing with PBS in column 3 and so on across the plate.

9. Incubate the plate at 37oC for 45 minutes.

10. Wash the plate 3x in wash buffer.

11. Aliquot 100µl anti-mouse HRP conjugate at 37oC for 30 minutes.

12. Wash plate 3x in wash buffer.

13. Aliquot 50µl TMBlue into each well of the microtitre plate.

14. Incubate at 37oC until colour development.

15. Read on plate reader at 630nm [Program 5].

 

2) Elisa for Antipeptide Sera

Phosphate Citrate Buffer:

0.025M citric acid (91.05g per 200ml)

0.05M disodium hydrogen phosphate (1.78g per 200ml)

 

Peroxidase substrate:

40mg OPDA in 50ml phosphate/citrate buffer

Add 50µl H2O2 immediately before use.

 

Method:

1. Add 100µl of 1% glutaraldehyde in PBS to each well and leave at least overnight at 4oC.

2. Remove glutaraldehyde and wash x1 with PBS.

3. Add peptide (50µl per well) at a concentration of 10µg.ml-1 in PBS and leave overnight at 4oC. Leave first column empty.

4. Remove peptide and dry plates at 37oC for 15 minutes.

5. Block with 150µl per well 10% Marvel in PBS at 37oC for 3 hours.

6. Wash 3x in 1% Marvel in PBS.

7. Make a doubling dilution series of antibody in a separate plate - 50µl final volume in 1% Marvel in PBS. Transfer to peptide plate and leave at room temperature for 1 hour.

8. Wash x3 in 1% Marvel in PBS.

9. Add 50µl of 1:500 dilution of second antibody in 1% Marvel in PBS and leave at room temperature for 1 hour. (2nd antibody is stored frozen as 50µl aliquots -50µl to 25ml PBS/Marvel).

10. Wash x3 with 1%Marvel in PBS and x2 in phosphate/citrate buffer.

11. Add 100µl OPDA reagent. Read on plate reader using a 492nm filter.

Adsorption with peptide

Make doubling dilution series of antibody using 100µg.ml-1 peptide in PBS as diluent

or

Make doubling dilution series starting at neat instead of 1/2 and then add 50µl of 100µg.ml-1 peptide in PBS to each well. Transfer 50µl to test plate.

ELECTROPORATION OF THP1 CELL LINES

• Split cells: 50 ml unfed cells + 150ml 10% FCS in RPM!-1640.

• Grow the cells for three days.

• Concentrate the cells to 10ml media and count.

• Use 2 x 107 cells per electroporation resuspended to 300µl in complete media containing 20mM Hepes.

• Add 20µg to cells to be electroporated (1µg DNA per 106 cells). Add the DNA to the cells in the centrifuge tube and mix well before transferring the mixture to a 4mm electroporation cuvette.

• Electroporation conditions are: 320V

1500µF

Maximum resistance

• Keep electroporation cells on ice 5 minutes before and after electroporation.

• Add electroporated cells to 20ml 10% FCS in RPMI-1640 media containing 20mM Hepes.

• Incubate 24 hours.

• Harvest cells and determine luciferase activity of each transfection.

Home-made ECL

Solution 1:

1 ml luminol, 0.44 ml coumaric acid, 10 ml 1 M Tris HCl pH 8.5

make up to 100 ml with distilled water

 

Solution 2:

64 microlitres 30% hydrogen peroxide, 10 ml I M Tris HCl pH 8.5

make up to 100 ml with distilled water

 

Store solutions in fridge, wrapped in aluminium foil to prevent light damage. We use for about 5 weeks, after which sensitivity may decrease (although they rarely last that long).

Luminol stock 250 mM in DMSO, keep in dark at -20.

Coumaric acid stock 90 mM in DMSO, keep in dark at -20

Luminol - Fluka cat. no. 09253 25 g £40.30

p-Coumaric acid - Sigma cat. no. C-9008 25 g £17.30

Hydrogen peroxide 30% solution from your usual supplier.

The stock solutions last for years at -20.

ECL method for developing Western blots

Author Kate (from James)

Project NF-kB

Date 1-9-93

Reference Amersham ECL Western blotting protocol booklet

Method (For mini-gel transfers, 8.5 cm x 6 cm PVDF membrane ['Sigma])

1 Block membrane (transferred proteins-side upwards) in 20 ml ECL buffer (PBS:5% dried skimmed milk:0.1% Tween 20). 30 min rocking, 20 oC.

2 Transfer to 50 ml red-capped tube (with nesco-film balance) containing suitable dilution of primary antisera in 2 ml ECL buffer (1:3000 for IkB and p50 antibodies).
1 h rocking on rollers, 20 oC.
(For @ IkB affinity-purified rabbit polyclonal [60 µg. ml-1], use 1/2000 dilution [1 µl in 2 ml]).

3 Rinse in ECL buffer and transfer to square petri dish.
Wash 2 x 15 min (20 oC) in ECL buffer (10-20 ml).

4 Return to red-capped tube and incubate with suitable dilution of secondary antisera in 2 ml ECL buffer.
30 min rocking on rollers, 20 oC.
(For detection of above 1o antisera, use affinity purified HrP-@ rabbit Ig Fab'2[Amersham] at 1/4000 [0.5 µl in 2 ml]).

5 Rinse in ECL buffer and transfer to square petri dish.
Wash (20 oC) 2 x 15 min ECL buffer (10-20 ml), 2 x 5 min PBS:0.1% Tween (10-20 ml).

6 In dark room, add 1 ml ECL Reagent 1 to 1 ml ECL Reagent 2 (both from Amersham) in a square petri dish.
Remove XS liquid from filter and immerse in above mixture (proteins uppermost), 1 min.

7 Remove XS liquid and place filter between 2 dry sheet of acetate, noting orientation of filter.
In the dark, expose filter to X-ray film for a suitable length of time, ensuring side containing transferred proteins against film. (eg 1 min. 5 min, 25 min. For shorter times, filter can be held against film, for longer times, an autorad cassette is used).

8 Develop film as normal.

Notes

 

Solutions

 

ECL buffer (made fresh)

PBS (500 ml)
Skimmed dried milk (St. Andrews University Central Catering) 5% (25 g)
Tween 20 (Sigma) 0.1% (0.5 ml)

Dissolve milk in PBS by boiling (µ wave, 5 min). Add Tween. Filter under vacuum.

DEAE-dextran transfections

SOLUTIONS

1. TS solution

For 1 litre

Tris HCl 25mM Mr 120 3g

NaCl 137mM Mr 58.44 8g

KCl 5mM Mr 74.5 0.372g

CaCl2 0.7mM Mr 147 0.102g

MgCl2 0.5mM Mr 203 0.101g

Adjust pH to 7.4, then add:

Na2HPO4.12H2O 0.6mM Mr 358 0.21g

Correct pH to 7.4, then autoclave or sterile filter.

2. DEAE Dextran

DEAE dextran 500 000: 20 mg/ml in water

Sterile filter before use.

 

METHOD

Day 1

Prepare cells for transfection at 0.5 x 106 cells/ml

Day 2

Wash cells in PBS

Prepare MIX of TS/Dextran (9.5ml TS solution + 0.5ml DEAE dextran solution).

Resuspend cells gently in TS (750ml/transfection point)

Distribute cells into 15 ml tubes (e.g. 107 cells per transfection)

Add plasmid DNA (about 5mg per 107 cells)

Add 750 ml of MIX TS/Dextran

Wait for 20 minutes at room temperature

Wash cells in PBS (at last 15 ml)

Spin down 1000 rpm 5 min

Resuspend cells in medium at 0.5 x 106 cells/ml

Culture O/N

Day 3

24 hours after transfection do experiment.

 

ChIPs Protocol

1) Chromatin Immunoprecipitation (ChIPs) Protocol (Farnham Lab)

This protocol is based upon protocols from Mark Biggin, Dave Allis and Richard Triesman plus a fair amount of trial and error. We have successfully used this protocol with NIH 3T3, Friend, HeLa, Raji and CHO cells. However, you may have to optimize conditions for your specific cell type.

Day 1

1. Add formaledehyde directly to tissue culture media to a final concentration of 1%. We generally use 2 x 107 cells per antibody per timepoint. Fewer cells can be used but usually results in a lower signal to noise ratio. Incubate adherent cells on a shaking platform and suspension cells on a stir plate for 10 minutes at room temperature. Crosslinking for longer periods of time (>30 minutes) tends to cause cells to form into a giant crosslinked aggregate that can not be efficiently sonicated.

2. Stop the crosslinking reaction by adding glycine to a final concentration of 0.125 M. Continue to rock or spin at room temp for 5 minutes.

3. For adherent cells, pour off media and rinse plates twice with cold 1X PBS. For suspension cells, centrifuge and wash cell pellet twice with cold 1X PBS.

4. Add an appropriate volume (we use 5 mls per 500 cm2 dish) of 1X PBS or a 20% trypsin solution in 1X PBS. Incubate at 370 C for 10 minutes if using trypsin (we have found this step useful for cells which are difficult to swell. Thus, for cell types that are easily swelled, this step may not be necessary).

5. Following addition of trypsin or PBS, scrape adherent cells from dishes. If you have used trypsin, inactivate the trypsin by adding a small amount of serum. Centrifuge scraped adherent or suspension cells and wash pellet once with 1X PBS plus PMSF (10 ul per ml).

6 Resuspend cell pellet in cell lysis buffer plus the protease inhibitors PMSF (10 ul per ml), aprotinin (1 ul per ml) and leupeptin (1 ul per ml). The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10 minutes. Cells can also be dounced on ice with a B dounce several times to aid in nuclei release.

7. Microfuge at 5,000 rpm for 5 minutes at 40 C to pellet the nuclei.

8. Resuspend nuclei in nuclei lysis buffer plus the same protease inhibitors as the cell lysis buffer. Incubate on ice for 10 minutes.

9. Sonicate chromatin to an average length of about 600 bp while keeping samples on ice (the time and number of pulses will vary depending on sonicator). Microfuge at 14,000 rmp for 10 minutes at 40 C. At this point, chromatin can be snap frozen in liquid nitrogen and stored at -700 C for up to several months.

10. Carefully remove the supernatant and transfer to a new tube. Preclear chromatin by adding blocked Staph A cells. Use 10-15 uls of preblocked Staph A cells for every 1 X 107 cells that you started with.

11. Incubate on a rotating platform at 40 C for 15 minutes, no longer. Microfuge at 14,000 rmp for 5 minutes.

12. Transfer supernatant to a clean tube and divide equally among your samples. Be sure to include a "no antibody" sample. We also include a "mock" samples which contains 1X dialysis buffer instead of chromatin (no antibody and mock are critical to control for nonspecific interactions and DNA contamination of Ip and wash solutions....the final output of this experiment is analyzed by PCR). Adjust the final volume of each sample with IP dilution buffer plus protease inhibitors if necessary. Samples volumes should be between 200 and 500 uls. Add 1 ug of antibody to each sample.

13. Incubate on the rotating platform at 40 C for at least 3 hours. Overnight is fine. If you are using monoclonal antibodies, add 1 ug of an appropriate secondary antibody and incubate for an additional 1 hour.

Day 2

14. Add 10 uls of blocked Staph A cells to each sample. Incubate on the rotating platform at room temp for 15 minutes, no longer.

15. Microfuge samples. Save the supernatant from the "no antibody" sample as "total input chromatin".

16. Wash pellets twice with 1.4 mls of 1X dialysis buffer and four times with 1.4 mls of IP buffer. For each wash, dissolve the pellet in 200 uls of buffer and use an additional 200 uls of buffer to wash the pipet tip. Add an additional 1 ml of buffer. For each wash, incubate samples on a rotating platform for 3 minutes then microfuge at 14,000 rmp for 3 minutes at room temp. Try to remove as much buffer as possible after each wash without aspirating the Staph A cells. Efficient washing is critical to reduce background.

17. After the last wash, microfuge and remove the last traces of buffer. Elute antibody/protein/DNA complexes by adding 150 uls of IP elution buffer. Shake on vortexer for at least 15 minutes at setting "vortex 3". Microfuge at 14,000 rpm for 3 minutes. Transfer supernatants to clean tubes. Repeat and combine both elutions in the same tube.

18. After the second elution, microfuge samples at 14,000 rpm for 5 minutes to remove any traces of Staph A cells. Transfer supernatants to clean tubes. Add 1 ul of high concentration RNase A (10 mg per ml) and 5M NaCl to a final concentration of 0.3 M. Incubate samples in the 670 waterbath for 4-5 hours to reverse formaldehyde crosslinks. Add 2 and a half volumes of ethanol and precipitate at -200 C overnight.

Day 3

19. Microfuge samples at 14,000 rpm for 15-20 minutes at 40 C. Respin and remove residual ethanol. Allow pellets to air dry completely.

20. Dissolve each pellet in 100 uls of TE. Add 25 uls of 5X PK buffer and 1.5 uls of proteinase K to each sample. The "total" sample will be gunky and may have to be dissolved in a larger volume. Incubate in 450 waterbath for 1-2 hours.

21. Add 175 uls of TE to each sample. Extract once with 300 uls of phenol/chloroform/ isoamyl alcohol and once with 300 ul chloroform/isoamyl alcohol. Total input samples may need to be extracted twice.

22. Add 30 uls of 5M NaCl, 5 ugs of tRNA and 5 ugs of glycogen to each sample. Mix well then add 750 uls of ethanol. Precipitate in -200 C freezer overnight.

Day 4

23. Microfuge samples at 14,000 rpm for 20 minutes at 40 C. Allow pellets to air dry. Resuspend DNA in water or TE and analyze by PCR. We generally resuspend in Ip's in 30 uls and then dilute the "total" sample an additional 300 fold and use 2-3 uls for each PCR reaction.

Solutions

Cell Lysis buffer

5 mM PIPES pH 8.0

85 mM KCL

0.5% NP40

protease inhibitors

Nuclei Lysis buffer

50 mM Tris-Cl pH 8.1

10 mM EDTA

1% SDS

protease inhibitors

IP Dilution buffer

0.01% SDS

1.1% Trition X 100

1.2 mM EDTA

16.7 mM Tris-Cl pH 8.1

167 mM NaCl

1X Dialysis buffer

2 mM EDTA

50 mM Tris-Cl pH 8.0

0.2 % Sarkosyl

IP Wash buffer

100 mM Tris-Cl pH 9 (8 for monoclonal antibodies)

500 mM LiCl

1% NP40

1% deoxycholic acid

Elution buffer

50 mM NaHCO3

1% SDS

5X PK buffer

50 mM Tris-Cl pH 7.5

25 mM EDTA

1.25% SDS

Protease Inhibitors

100 mM PMSF in ethanol, use at 1:100

10 mg per ml in 0.01 M HEPES pH 8.0, use at 1:1,000

10 mg per ml leupeptin in water, use at 1:1,000

Staph A Cells

Resuspend 1 gram of lyophilized Staph A cells (Boehringer Mannheim) in 10 mls of 1X dialysis buffer. Centifuge at 10,000 rpm for 5 minutes at 40 C. Repeat. Resuspend in 3 mls of 1X PBS plus 3% SDS and 10% BME. Boil for 30 minutes. Centifuge at 10,000 rpm for 5 minutes. Wash in 1X dialysis buffer and cetrifuge at 10,000 rpm for 5 minutes. Repeat. Resuspend in 4 mls of 1X dialysis buffer. Divide into 100 ul aliquots, snap freeze and store in liquid nitrogen.

To block Staph A cells

Thaw 1 tube (100 uls) of cells for approximately every 108 cells that you begin with. Add 10 of herring sperm DNA (10 mg/ml) and 10 uls of BSA (10 mg/ml) to each tube of Staph A cells. Incubate on the rotating platform at 40 C for at least 3 hours, overnight is fine. Before using, microfuge for 3 minutes. Remove supernatant and wash pellet twice in 1X dialysis buffer. Resuspend cells in a volume of 1X dialysis equal to the original starting volume.

 

2) ChIP PROTOCOL (Olivier Geneste)

Protocol: Olivier Geneste's adaptation of Braunstein and coworkers' method.

References:

Braunstein et al, Genes and Devt 7, 592-604 (1993)

Meluh and Koshland, Genes and Devt 11, 3401-3412 (1997)

Alberts et al. Cell 92, 475 (1998).

 

3T3 cells from a 9cm dish ( 5 x 105 to 1 x 106cells) are crosslinked by addition of HCHO to the medium (1% final) for 10 minutes in incubator at 37C. (This is fine for Allis "penta" acetyl-H4 a/b but for other a/bs, crosslinking at 4C for 60min may be better). Aspirate medium, wash and scrape cells with cold PBS containing protease inhibitors, pellet by centrifugation.

The cell pellet is resuspended in 200µl of NP40 containing Buffer A / NP40 (5mM Pipes pH 8.0, 85mM KCl, 0.5%NP40, and protease inhibitors) and placed on ice for 10min. A crude "nuclei" fraction is pelleted by centrifugation (microcentrifuge, 5 min, 5krpm). The pellet is washed once in Buffer A without NP40, resuspended in 200µl of lysis buffer B (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) and incubated 10min on ice.

Lysed nuclei are sonicated under conditions that reduce DNA length to between 200 and 1000 basepairs. This is usually achieved by sonicating five times 10sec at the third of max power of our apparatus (Soniprep 150 MSE; Check the size of the sonicated DNA by gel electrophoresis after reversion of cross-links). After sonication debris is removed by centrifugation (10min, 13krpm, microfuge).

The supernatant is diluted tenfold in IP buffer (0.01% SDS, 1.1% tritonX100, 1.2mM EDTA, 16.7mM Tris pH8.1, 167mM NaCl, and protease inhibitors) to give the Chromatin Solution. Keep a part of it to check the input of material you had in different samples before the IP.

Preclear the chromatin solution (2ml) on protein A sepharose preadsorbed with sonicated salmon sperm DNA (100ml of protein A sepharose / 20mg sonicated salmon sperm DNA in TE, 1mg/ml BSA); 45' at 4oC on a rotating wheel). This preclearing step is essential to reduce background with nonspecific antibodies.

 

The beads are pelleted by centrifugation (20 sec in microcentrifuge), collect supernatant.

Incubate precleared chromatin solution (1ml) with antibodies for 2 hours to overnight (depending of the antibodies) at 40C on the wheel. Collect immune complexes with protein A beads preadsorbed with sonicated salmon sperm DNA (30 µl of Protein A sepharose as above).

Pellet beads by centrifugation and wash five times (1 ml wash, 3-5 min each):

1) 0.1 % SDS, 1 % tritonX100, 2 mM EDTA, 20 mM Tris-HCl pH8.1, 150 mM NaCl

2) 0.1 % SDS, 1% TritonX100, 2 mM EDTA, 20 mM TrisHCl pH8.1, 500 mM NaCl. (Note that this high salt wash may not be suitable for all the antibodies)

3) 0.25 M LiCl, 1 % NP40, 1 % deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.1.

4) TE pH8.

5) TE pH8.

Elute immune complexes by adding 0.25ml 1% SDS / 0.1 M NaHCO3. Vortex briefly, incubate 15 min at room temperature on wheel. Spin down, collect the supernatant. Repeat elution and combine eluates.

Add 20ml 5 M NaCl to the 500ml of eluted material, reverse crosslinks at 650C, 4hr. Add 10ml of 0.5 M EDTA, 20 ml of 1 M Tris-HCl pH6.5 (to buffer the solution) and 2 ml proteinase K (10 mg/ml), incubate 1 hour at 450C.

Recover DNA by phenol extraction and ethanol precipitation. Detect specific sequences by PCR or slot-blot.

BUFFERS 

A. Lysis buffer:

Reagent	Concentration	Amount	Final Concentration
Sodium fluoride		2.1g	50mM
Tetra-sodium pyrophsophate		2.26g	5mM
Sodium orthovanadate		0.185g	1mM
ß-glyceropyro- phosphate		0.215g	10mM
Nonidet P40		5ml	0.5%
EDTA	500mM	4ml	2mM
Na2HPO4	200mM	84ml	20mM
NaH2PO4	200mM	16ml	20mM
Distilled water		to 1000ml

For nucleoplasmic fractions add 85µl 5M NaCl per ml of lysis buffer.

B. RIPA buffer:

Reagent	Concentration	Amount	Final Concentration
NaCl	5M	5ml	50mM
Tris, pH 8.2	1M	12.5ml	25mM
Nonidet P40		2.5ml	0.5%
Deoxycholate		2.5g	0.5%
SDS	25%	2ml	0.1%
Azide		0.5g	0.1%
Distilled water		to 500ml

 

C. Protease Inhibitors:

To 1ml. Lysis buffer add:

Protease Inhibitor	Concentration	Volume/µl	Final Concentration
Leupeptin	1mM	1µl	1µM
Pepstatin	1mM	1µl	1µM
Bestatin	5mg.ml.-1	8µl	40µg.µl.-1
Pefabloc	100mM	10µl	1mM
TPCK	1mM	20µl	20µM

Lysis buffer - phosphatase inhibitors
20 mM sodium phosphate buffer pH 7.5
50 mM NaF
10 mM Na b-glycerophosphate
5 mM Na pyrophosphate
1 mM Na orthovanadate
1 mM EDTA
1 mM EGTA
1 mM DTT
0.1 mM OKA
0.5% NP40
Add protease inhibitors just before use
Spin down cells
wash x1 with ice cold PBS
Disperse pellet of cells by vortexing
Add lysis buffer (approx 4 volumes) and quickly resuspend cells
Spin for 10 sec. in a microfuge
Remove SN ---> cytoplasm.
Disperse pellet of nuclei by vortexing
Add lysis buffer (approx 4 volumes- of nuclei, usually about half of above) and quickly resuspend nuclei
Determine the exact volume and add 5M NaCl to 0.3-0.4M (depending on cells).
Mix quickly and leave on a rotating wheel in the cold room for 30'
Spin on a refigerated microfuge for 5'
Remove SN ---> nuclear extract.
If you are really worried about getting clean nuclei then disperse the cells in a lage volume of lysis buffer eg 50 ml and then spin out the nuclei eg 2.5 k for 5' Take up the nuclei in a small volume and proceed as above.

MAINTENANCE OF CELL LINES
The following table gives details of cell lines used and maintenance conditions.

Cell line	Cell type	% FCS	% HI. FCS	Media	Split
Daudi	B-cell	10	-	RPMI-1640	1:5
Jurkatt	T-Cell	5	-	RPMI-1640	1:5
THP1	Monocyte	10	-	RPMI-1640	1:5
U937	Monocyte	5	-	RPMI-1640	1:5
HDLM2	Mixed lymphocyte	-	20	RPMI-1640	1:5
KMH2	Mixed lymphocyte	-	10	RPMI-1640	1:5
L428	Mixed lymphocyte	-	10	RPMI-1640	1:5
COS7		10	-	GMEM	1:10
HELA		10	-	GMEM	1:10
AUV-A549		10	-	GMEM	1:10
a 5.1	T-cell	10	-	RPMI-1640	1:10

* a 5.1 is maintained in media supplemented with 200m g ml-1 geneticin.
FCS: foetal calf serum
HI. FCS: heat inactivated foetal calf serum

To find SUMO Consensus Motifs in the proteins click here

To find SUMO Interactive Motifs (SIM) in the proteins click here

To find RING motifs in the proteins click here

To find PolyQ/PolyP motifs in the proteins click here

To find Poly Sumo Binding Motif in the proteins click here