Discontinuous SDS Polyacrylamide gel electrophoresis. *
Stock solutions; *
Preparation *
HIGH RESOLUTION TRIS-TRICINE SDS GELS *
Solutions *
Glycerol Gels *
Mini-gel Recipe *
Fluorography *
Western Blotting *
western probing *
Western Blot Stringent Washing Procedure. *
Footprinting *
Buffers:- *
Binding Reaction:- *
Extrait Dignam modifié *
Solution C: *
Solution D: *
Solution C/20: *
Discontinuous SDS Polyacrylamide gel electrophoresis.
Acrylamide solution (30%:0.8% bis)
1.5M Tris.HCl pH 8.8, 0.4% SDS (Buffer A/ Lower)
0.5M Tris.HCl pH 6.8, 0.4% SDS (BufferB/Upper)
4X Sample LB;
250mM Tris.HCl pH 6.8,
2% (w/v) SDS,
20% (v/v) 2-mercaptoEtOH,
40% (v/v) glycerol,
1% (w/v) BPB.
5X Running Buffer;
|
250mM Tris base |
60g |
|
1M glycine, |
150g |
|
0.5% (w/v) SDS |
10g |
|
___ |
|
|
Final Volume |
2l |
10% Ammonium Persulphate, made freshly.
Comassie blue staining solution;
|
0.125% Comassie brilliant blue R-250 |
1.3g |
|
50% methanol |
500 mL |
|
10% acetic acid |
100 mL |
|
____ |
|
|
final volume |
1l |
Destaining solution; 20% methanol, 10% acetic acid (can use 50% or more but may destain protein bands)
Running Gel:-
For a general analytical gel use 12% acrylamide. 15ml resolving gel is more than enough for two 0.75 mm minigels, e.g. Protean minigel;
|
For 15ml resolv. gel:- |
5% |
10% |
12% |
15% |
|
Acrylamide soln. 40% or Acrylamide soln. 30% |
1.83ml 2.5ml |
3.75ml 5ml |
4.5ml 6ml |
5.7ml 7.5ml |
|
Buffer A |
3.75ml |
3.75ml |
3.75ml |
3.75ml |
|
Distilled Water; 40% acryl. or ; 30% acryl. |
9.42ml 8.75ml |
7.5ml 6.25ml |
6.75ml 5.25ml |
5.55ml 3.75ml |
|
10% Amm. Persulphate |
50ul |
50ul |
50ul |
50ul |
(Solution may or may not be degassed at this point, but if it is use a very large erlenmeyer.)
|
TEMED |
10ul |
10ul |
10ul |
10ul |
Note: gradient minigels 0.75mm thick can be made using a gradient mixer. Take 2ml of 5% gel solution in first chamber and 2 ml of 15% in second each with 20ul of 10% persulphate and 2ul TEMED in each. Pump into gel and immediately wash the gradient maker and pump through with water to prevent acrylamide polymererising in them.
Overlay resolving gel with water-saturated butan-1-ol (n-Butanol) until polymerised. Then wash top of gel well with distilled water or resolving buffer and pour stacking gel:-
Stacking gel:-
For 5ml of stacking gel;
|
Acrylamide soln. 40% or Acrylamide soln. 30% |
0.48ml 0.65ml |
|
Buffer A |
1.25ml |
|
Distilled Water |
3.05ml |
|
10% Amm. Persulphate |
25ul |
(Solution may or may not be degassed at this point, but if it is use a very large erlenmeyer.)
|
TEMED |
5ul |
Make samples to 1x sample loading buffer and place in boiling water bath for 5 min..
Electrophorese minigels (~ 10cm long) at 150V constant voltage for about 1hr or 20cm gels at 300V (water cooling) for several hrs or at 45V overnight.
HIGH RESOLUTION TRIS-TRICINE SDS GELS
(Excellent for proteins less than 10 kD)
Resolving gel
4XTris-SDS for the resolving gel:
1M Tris-HCl, pH 8.45. 0.4% SDS
Stacking gel
4XTris-SDS for the stacking gel:
0.5 M Tris-HCl, pH 6.8 (same as for an ordinary Tris-glycine SDS gel)
Acrylamide solution
30% Acrylamide:0.8% bis-acrylamide (same as for an ordinary Tris-glycine SDS gel)
Running buffer
|
5x |
1x |
|
|
500 mM Tris base |
60.5g |
12.1g |
|
100mM Tricine |
89.6g |
0.9g |
|
0.1% SDS |
5.0g |
1.0g |
|
____ |
____ |
|
|
final volume |
1l |
1l |
The pH of this buffer will be 8.25 (not necessary to adjust)
Best results are obtained with an 7.5-20% gradient gel. This recipe is for 0.75mm thick mini-protean (BioRad) gels.
Prepare 20-30 ml 7.5% and 20% resolving gel solutions without catalysts. Keep these at 4oC or even R/T. Prior to pouring the gel, take 2 ml of each vial and add 13ul APS and 1.3ul TEMED (Do this on ice). Put the gel solutions in a gradient maker (the 20% solution in the front chamber, the 7.5% in the back one. A magnetic stirrer bar is needed only for the front chamber. The gradient maker must be connected to a peristaltic pump and the gradient made by a speed of circa 4ml per minute. To the other end of the pump tubing attach a precut sequencing gel tip and this is inserted between the gel plates. After pouring the gel the gradient maker and the pump must be washed immediately by running the whole equipment with distilled water. Overlayer the resolving gel with water-saturated until polymerization. After removing the butanol a 4% stacking gel is poured (as for standard tris-glycine SDS gels). The sample buffer is identical to that for the tris-glycine gels.
Run the gel at 10mA until the samples reach the resolving gel border, then increase the voltage, but do not exceed 30 mA.
(method of Perrie and Perry (1970) Biochem J 119:31)
|
|
0.75 mm gel |
1.5 mm gel |
|
30% acrylamide (10% final) |
2.0 ml |
4.0 ml |
|
0.2M Tris/glycine |
0.6 ml |
1.2 ml |
|
Glycerol (40% final) |
2.4 ml |
4.8 ml |
|
H20 |
1.0 ml |
2.0 ml |
|
Temed |
18 ul |
36 ul |
|
10% APS |
6 ul |
12 ul |
0.2 M Tris/glycine
12.11 g Tris (0.2M final conc.)
8.63 g glycine (0.23M final conc.)
Final volume 500 ml, pH ~8.6
Sample Preparation
For wet samples: Add dry urea until saturation is acheived. You are hoping for a final concentration of 8 M urea. Per 50 ul of sample add 1 ul 0.5 mg/ml bromphenol blue and 2 ul of 1 M DTT/0.2 M EDTA. Do not boil the samples!
For WC lysates: Add ice-cold 10% TCA, 10 mM DTT to cell monolayer and freeze cells by placing dish on dry ice. Scrape cell monolayer into 10% TCA, 10 mM DTT while on dry ice. Incubate extract for 20 min on wet ice and collect insoluble material by spinning at 4700 X g for 1 min. Wash the pellets 3 times with acetone and air dry to remove residual acetone. Resusupend pellets in 8 M urea, 20 mM Tris base pH 8.6, 22 mM glycine, 10 mM DTT and 0.004% bromphenol blue.
Electrophoresis
Running buffer: Use a 1:10 dilution of the 0.2 M Tris/glycine
Prior to sample loading rinse wells very well. Run the gel at 350 V. After the dye has run off continue to run the gel for 5-15 minutes.
A. Bannister
1. Fix gel in fixer, 30% (v/v) methanol, 10% (v/v) acetic acid for at least 1 hour.
2. Submerge in glacial acetic acid for 15 min..
3. Immerse in 200ml of 20% (w/v) PPO in glacial acetic acid for 90 min..
4. Place in 500ml deionised water for 30 min..
5. Place in 200ml 30% (v/v) methanol, 3% (v/v) glycerol for 60 min..
6. Dry on gel drier, 60 oC for 2-3 hours.
Transfer Buffer:
|
Tris-base |
12.1g |
|
Glycine |
57.8g |
|
Methanol |
800ml |
|
___ |
|
|
final volume |
4 liters |
Ponceau Rouge:
|
0.2% Ponceau Red, |
1% CH3 COOH |
India Ink:
0.1% India ink
0.2% Triton X-100
1 x PBS
1. Cut four squares of 3MM paper to the size of the Scoth-Brite sponge pads and one cut piece of nitrocellulose membrane (Hybond-C or similar) to the size of the gel or a little larger. Note; do not touch filter with the hands.
2. Submerge Scotch-Brite sponges in transfer buffer in a glass dish and remove bubbles
3. Place the open gel holder in the dish and position one of the sponges against the "black" side of the holder.
4. Wet two squares of 3MM paper in transfer buffer and place these on the sponge making sure that there are no bubbles caught between the sponge and the paper or between the sheets.
5. Wet the 3MM papers with fresh transfer buffer and place the gel onto them, again assuring that no bubbles are trapped.
6. Wet the gel with tranfer buffer and place the prewetted membrane on to the gel, taking care to avoid trapped bubbles.
7. Place two more prewetted 3MM squares onto the membrane followed by the second buffer saturated sponge, avoid trapped bubbles.
8. Close the gray side of the gel holder down onto the gel sandwich, click shut and rapidly place (submerge), hinge side up, in the buffer filled transfer tank, black gel support facing the (black, negative) anode.
9. Transfer with cooling (very slow flow of tap water or transfer in 4oC room) O/N at 20 to 30V or (with greater cooling) at 70V for 1 to 2 hr. These conditions are appropriate for minigel tanks of around 10 cm between electrodes. Note; two gels can generally be transfered in the same tank but this often causes some loss of gel resolution on the tranfer. Never be tempted to transfer more than two gels at a time, despite the fact that most tanks have positions for up to four gels at a time.
10. Remove the gel from the sandwich and colour for a few minutes with the solution of ponceau rouge or in India Ink for about 15-30 min. before probing
1 Incubate the blot in 5% skimmed milk, 0.2% Tween, 1xPBS for about 15-30 min.
2 Incubate with the first antibody in PBS-T: 1xPBS, 0.2% Tween, 1% skimmed milk for 1 h at room temperature
3 Wash the blot 3-4 X 10 min in PBS-T:
4 Incubate the blot in second antibody (peroxidase conjugated) for 45 min. The second antibody should be diluted 1: 5-8000 in PBS-T
5 Wash 3-4X PBS-T
6 Reveal with ECL reagents (Generally equal amounts of both solutions)
Western Blot Stringent Washing Procedure.
Stefan Dimitrov 1994
1. After the blotting, incubate the blot in 0.1% India ink, 0.2% Triton X-100, 1xPBS for about 15-30 min.
2. Rinse the blot with distilled water, and incubate it with the first antibody for 1 h at room temperature under gentle shaking. The first antibody is recommended to be in a concentration of 1ug/ml in a solution of 10% calf serum, 0.2% Tween 20, 1xPBS, 0.02% sodium azide(the prepared antibody in this way could be kept at 4oC for months without noticeable loss of activity and reused many times)
Important: it is recommended to work with immunopurified antibody
3. Rinse very quickly(for no more of 5 sec) the blot with distilled water
4. Wash the blot for 15 min in buffer (A): 1 x PBS, 0.5% Triton-X100, 0.5M NaCl under shaking
5. Repeat step 4, but for 5 min
6. Wash the blot in buffer (B): 1xPBS, 0.5% Triton X-100 for 15 min under shaking
7. Repeat step 6, but for 5 min only
8. Repeat step 3
9. Incubate the blot in second antibody (peroxidase conjugated) for 45 min under shaking at room temperature. The second antibody should be diluted 1: 3-4000 in 1% milk, 1xPBS, 0.2% Tween-20.
10. Repeat steps 3 to 8.
11. Reveal with ECL
N.B. a)The rinsing with distilled water is very important since in this way the background is very efficiently eliminate
b) I recommend highly an immunopurified antibody be used.
2x reaction buffer (no KCl)
|
final concentration |
||
|
glycerol, 100%, |
2ml |
20% |
|
0.5M EDTA, |
4ul |
0.2mM |
|
1M DTT, |
10ul |
1mM |
|
1M HEPES, pH 7.9, |
200ul |
20mM |
|
8% PVA, (takes O/N) |
5ml |
4% |
|
____ |
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|
finale volume |
10ml |
poly dA-T:-
1mg/ml (Pharmacia) in 150mM KCl.
DNAase Buffer:-
10mM MgCl2,
5mM CaCl2
Stop Buffer:-
1% SDS,
0.2M NaCl,
20mM EDTA
tRNA:- 50mg/ml in TE
25 ul of 2x reaction buffer, 0.5 ul polydA-T, protein solution (10ul or more), add H2O to 47 ul adjusting to 80 to 100mM KCl. Add 3 ul probe (15,000 cpm, <3ng), incubate 15 min. 0 oC. Add 50 ul DNAase buffer, 5 ul DNAase I (conc. 0.05mg/ml for crude extracts to 5ugm/ml for semi-pure and 0.1 ugm/ml for naked DNA, dil. in H2O from 5mg/ml stock in ?), incubate 2 min. at R/T. Stop reaction with 100ul stop buffer, add 5ugm tRNA. Phenol/chloroform extract and prec.
Les cellules N1-S1 cultivées en spinner (RPMI-1640 + 10% FBS) sont récoltées à environ 750 000 cell/ml. dans des bouteilles de 250 ml remplies au 3/4 à 5300 RPM pendant 4 min. dans un rotor GSA. (dépendement du volume on répète autant de fois que nécessaire.)
On lave trois fois les cellules dans PBS (0,5 mM DTT+PMSF) pour finir avec le culot dans une seule bouteille (2L nécessaire). Centrifugation 4 min. à 5300 RPM dans GSA. On resuspend les cellules en ajoutant un petit volume de PBS, on frappe la bouteille contre la paume de la main et on remet du PBS.
On transfère le culot dans un tube à fond conique (centrifugation 1000 RPM, 2 min. dans R3C3) et on mesure le volume de cellules. Lorsque l'on reparlera du volume de cellule, c'est toujours à celui-ci que l'on réfère. On resuspend les cellules dans 5 vol. de solution A et on laisse gonfler sur glace 10 min. On inverse 2 fois avant de centrifuger 10 min. à 2000 RPM dans RC3, on aspire le surnageant et on resuspend dans 2 vol. de solution A.
Homogénéisation: Avec Dounce et piston B, on donne de 12 à 20 coups et on vérifie la lyse au microscope (pas évident). On centrifuge 10 min à 2000 RPM dans RC3 on enlève le surnageant et on recentrifuge le culot 20 min. à 16 500 RPM dans SS-34.
On resuspend le culot dans 1,5 vol. de solution C, on mélange un peu avec le piston du Dounce (ce culot devrait être plus visqueux que le culot de cellules et se défaire difficilement) et on mélange avec agitateur magnétique pour 30 min. on centrifuge ensuite 30 min. à 16 500 RPM dans SS-34. On dialyse ensuite le surnageant contre 50 vol. de solution D pendant deux heures.
*On centrifuge le dialysat 20 min. à 14500 RPM dans SS-34, on mesure le volume de surnageant et on ajoute graduellement 0,472g Ammonium sulfate par ml de surnageant. on agite sur agitateur pendant 30 min. On centrifuge 20 min. à 18 000 RPM dans SS-34 et on resuspend le culot dans 1/3 de volume de C/20. On dialyse contre C/20 (2 hres ou O/N) on centrifuge 20 min à 14 500 RPM dans SS-34 et on congèle les aliquotes.
Solution A: (toutes les solutions sont dans 0,5 mM DTT + PMSF)
10 mM HEPES pH.:7,9
1,5 mM MgCl2
10 mM KCl
20 mM HEPES pH.:7,9
25% glycérol
0,42 M NaCl
1,5 mM MgCl2
0,2 mM EDTA
20 mM HEPES pH.:7,9
20% glycérol
0,1 M KCl
0,2 mM EDTA
20 mM HEPES pH.:7,9
25% glycérol
0,1 M KCl
5 mM MgCl2
0,2 mM EDTA