此项技术是一种蛋白质的固定和分析技术,是将已用聚丙烯酰胺凝胶或其它凝胶或电泳分离的蛋白质转移到硝酸纤维滤膜上,固定在滤膜上的蛋白质成分仍保留抗原活性及与其它大分子特异性结合的能力,所以能与特异性抗体或核酸结合,其程序Sonthern Blot相似,故称为Western Blot,第一抗体与膜上特异抗原结合后,再用标记的二抗(同位素或非同位素的酶)来检测,此方法可检测1ng抗原蛋白。
1、配液:
(1)转移电泳缓冲液:20mmol/L Tris HCL pH8.0
150mmol/L 甘氨酸
加14.5g Tris粉 67.08g甘氨酸于4L水中,加入1200mL
甲醇,加水至6L
(2)丽春红S溶液:0.5%丽春红S
1%乙酸
2、操作步骤:
(1)用已制备好的SDS-PAGE分离的蛋白凝胶。
(2)用一张滤纸,剪成与胶同样大小,在转移电泳缓冲液中预湿,放在Scotch-Brit Pad上,在胶的阴性端放上滤纸,胶的表面用该缓冲液浸湿,排出所有气泡。
(3)在胶的阳极面放置同样大小浸湿的硝酸纤维素膜,排出气泡,再在滤膜的阳极端放置一张滤纸,排出气泡,再放一个Scotch-Brit Pad。
(4)将以上“三明治”样装置放入一个塑料支撑物中间,将支撑物放入电转移装置中,加入电转移缓冲液。
(5)接通电源:使胶上的蛋白转移到硝酸纤维素膜上,电压为14V 4℃转移4小时或过夜。
(6)将滤膜放入丽春红S溶液中5分钟,蛋白染色水中脱色2分钟,照像,用印度墨水将分子量标准染色,在水中完全脱色。
(7)将滤膜放在塑料袋中,每3张加入5mL封闭缓冲液(1克速溶去脂奶粉溶于100ml PBS中),封闭特异性抗体结合位点,室温1h,摇动,倒出封闭缓冲液。
(8)在封闭缓冲液中稀释第一抗体,加入后室温放置1小时,将滤膜转到塑料盒中,用200mL PBS洗四次,摇动。
(9)在封闭缓冲液中稀释辣根过氧化物酶标记的二抗,重复步骤8。
(10)将滤膜放在100mL新配制的DAB底物溶液中,大约2~3分钟就可显色,用水冲洗终止反应、照像。
结果分析:分析阳性(显色)条带的分子量大小,而且根据信号(颜色)强弱分析蛋白表达量。
For 10% SDS-PAGE:
1) clean plates with ethanol
2) set-up apparatus
3) make resolving gel mix:
~5 mL
water 1.9 mL
30% polyacrylamide (29:1) 1.7 mL
1.5 M Tris pH 8.8 1.3 mL
10% SDS 50 L
10% APS 50 L
temed 8 L
4) pour gel at angle
5) use 0.01% SDS to keep top of gel even
6) allow to polymerize
7) get rid of 0.01% SDS with blot paper
8) make stacking gel mix:
~1 mL
water 680 L
30% polyacrylamide (29:1) 170 L
1 M Tris pH 6.8 130 L
10% SDS 10 L
10% APS 10 L
temed 10 L
9) pour stacking gel, add gel comb
10) allow to polymerize
11) add 2x SDS-PAGE sample buffer to samples
12) boil samples 5’
13) load onto gel with marker
14) run gel in Tris-Glycine (w/ SDS) Electrophoresis Buffer at 100 V about 60’
For Western blot:
10x Towbin Buffer: 30.3 g 25 mM Tris
144 g 192 M glycine
(w/ or w/o 100 mL 10% SDS)
adjust volume to 1 L w/ ddH2O
1x Towbin Buffer: 100 mL 10x Towbin Buffer
200 mL methanol
adjust volume to 1 L w/ ddH2O
1) cut nitrocellulose
2) soak gel (w/o stacking gel) in Towbin Buffer 15’
3) soak nitrocellulose in Towbin Buffer 15’ (put in at 45 degree angle to prevent bubbles)
For semi-dry Western blot:
1) wet extra thick blot paper in Towbin Buffer
2) set-up Western: ---------------------------------- extra thick blot paper
####################### gel
++++++++++++++++++++ nitrocellulose
---------------------------------- extra thick blot paper
3) while setting-up stack, use tube or pipet to prevent air bubbles between layers
4) transfer protein to membrane: 25 V for 60’ (1 minigel) (manual gives different directions)
For wet Western blot:
1) wet normal blot paper and sponges in Towbin Buffer
2) set up Western: @@@@@@@@@@@@ clear plastic side
~~~~~~~~~~~~~~~~~~~~~ sponge
---------------------------------- blot paper
++++++++++++++++++++ nitrocellulose
####################### gel
---------------------------------- blot paper
~~~~~~~~~~~~~~~~~~~~~ sponge
@@@@@@@@@@@@ black plastic side
3) while setting-up stack, use tube or pipet to prevent air bubbles between layers
4) set-up apparatus
5) place ice in apparatus
6) fill up apparatus with Towin Buffer (w/o touching ice)
7) transfer protein to membrane: 100 V for 60’ (1 minigel)
For antibody probing:
TrisHCl/NaCl: 0 mL 1 M TrisHCl pH 7.5
30 mL 5 M NaCl
fill to 1 L with water
5% TrisHCl/NaCl/milk: 100 mL TrisHCl/NaCl
5 g nonfat dry milk
0.5% TrisHCl/NaCl/milk: 100 mL 5% TrisHCl/NaCl/milk
900 mL TrisHCl/NaCl
1) block membrane in 5% TrisHCl/NaCl/milk for at least 1 hour
2) dilute block buffer in remaining TrisHCl/NaCl
3) prepare 1st antibody dilution in diluted blocking solution
4) incubate with 1st antibody at least 1 hour to O/N
5) wash with diluted blocking solution 3x (or 2x) for about 10’
6) prepare 2nd antibody dilution in diluted blocking solution
7) incubate with 2nd antibody (HRP conj) at least 1 hour to O/N
8) wash with diluted blocking solution 3x (or 2x) for about 10’ [1:50,000 dilution of 1:2 1:100,000 final dilution]
9) mix equal parts luminal/enhancer solution and stable peroxide solution
10) incubate for about 5’
11) expose/develop film
12) can store membranes wrapped in saran (and foil) at -20°C or 4°C
Western Blotting SOP
1. Separate proteins by SDS-PAGE. Include prestained molecular weight standards in one or more gel lanes: PageRuler Prestained Protein Ladder (Fermentas cat# SM0671)
Always use clean gloves when handling gels and membranes. Oil from hands blocks the transfer.
2. Prepare transfer membrane. Nitrocellulose membrane pore size 0.45 m is for proteins >15 kDa. For proteins <15 kDa, use 0.2 m nitrocellulose. If using nitrocellulose from a roll, cut it to the same size as the gel plus 1 to 2 mm each edge. Place into MQ water slowly, with one edge at a 45 angle. The water will wick up into the membrane, wetting the entire surface. If it is inserted too quickly into the water, air gets trapped and will appear as white blotches in the membrane. Protein will not transfer onto these areas. Once wet, equilibrate the membrane in transfer buffer for 15 min.
3. When SDS-PAGE is complete, disassemble the gel sandwich and remove the stacking gel. Briefly equilibrate the gel in transfer buffer for 4-5 min (but not longer).
4. Assemble the transfer sandwich in a tray large enough to hold the plastic transfer cassette. Fill with transfer buffer so that the cassette is covered. The black side should be down, submerged to start the sandwich. The transfer cassette should be assembled under buffer to minimize trapping of air bubbles.
5. On bottom half of the plastic transfer cassette, place a Scotch-Brite pad, followed by a piece of filter paper prewet with transfer buffer.
6. Place the gel on top of filter paper. Remove any air bubbles between gel and filter paper by gently rolling a test tube or glass rod over the surface of the gel. Protein will not transfer where there are air bubbles.
7. Moisten surface of the gel with transfer buffer. Place prewetted membrane directly on top side of gel and remove all air bubbles as in step 6. Some proteins will transfer as soon as the gel is placed on the membrane; repositioning the gel or membrane can result in a smeared or double image on the developed blot.
8. Wet another piece of filter paper, place over the membrane and remove all air bubbles. Place a Scotch-Brite pad on top.
9. Complete the assembly by locking the top half of the transfer cassette into place (white side).
10. Fill the tank with transfer buffer and place transfer cassette containing sandwich into electroblotting apparatus in correct orientation (double-check this!). In the coldroom, connect the leads of the power supply to the corresponding sides of the electroblotting apparatus.
11. Run at 350 mA constant current for 1 hour or overnight at 30 V (constant voltage). Put the tank on a stir plate and insert a stir bar. Stirring is important for preventing hot spots in the tank. For 30 V overnight constant voltage, the current stays within acceptable limits (30 to 70 mA ) and transfer is near complete. Always insert a cold pack if you do the high current 1 hour transfer.
12. When transfer is complete, remove membrane and mark the orientation by cutting the upper right corner. The membrane can also be marked with an HB pencil (not pen). Stain the membrane in coomassie blue as usual to check for untransferred proteins.
13. Optional: If you will air-dry the membrane, rinse it 3 times for 5 min each in fresh changes of MQ water.
14. At this point, the membrane can be air-dried (about 1 hour on filter paper at room temp), wrapped, and stored at -20C until immunoblotting.
15. Check efficiency of transfer by staining with Ponceau S: If the membrane was stored dry, wet the membrane in MQ water as in step 2. Place the membrane in Ponceau S solution for 5 min at room temperature.
16. Destain 2 min in water and scan the membrane if desired.
17. Completely destain the membrane by soaking an additional 10 min in a large volume of water.
18. Block the membrane in 5% fat-free milk powder (see below) for 1 hour at room temperature with gentle rocking. This can be down overnight at 4C.
19. Rinse the membrane briefly in two changes of TBS-T.
The next steps will depend on whether you have antibody linked to horse radish peroxidase (HRP; e.g. anti 6-his-HRP requires just this one antibody, see below) or if you use an unlabeled primary antibody together with an HRP-labeled secondary antibody.
20. Incubate the membrane in primary antibody for 1 hour at room temperature, diluted in TBS-T. Make sure the membrane is completely covered and will not dry out. Use a minimal volume of antibody solution (3-4 ml per blot). This is best done using a heat-sealable pouch.
21. Rinse briefly in TBS-T, and then wash in a large volume of TBS-T:
Once for 15 min.
3 times for 5 min. each in fresh TBS-T.
22. Incubate the membrane in secondary antibody for 1 hour at room temperature, diluted in TBS-T (usually a 1:5000 dilution). Again, the best way is in a heat-sealable pouch.
23. Wash the same as for the primary antibody, in step 21.
24. Place the blots in a container with a lid in TBS (no tween) and rinse briefly.
25. Bring the following with you to the processor room in Biological Sciences (room G5-09).
Film
Your blots
Metal film cassette (in case a long exposure is required)
Blot holder (with luminescent paint orientation marks)
5 ml pipet and tips
Detection reagent
Parafilm-covered plexiglass for detection
A marker
Timer
Forceps
25. Mix each reagent in a 1:1 ratio. A final volume of 0.125 ml/cm2 membrane is required (about 3 ml for a full-size blot). Just pipet the volume required directly onto the parafilm and mix carefully.
26. Add the blots and incubate for 1 min (protein side up). Agitate gently and ensure the entire surface is covered with solution.
27. Drain excess reagent.
28. Insert blots into blot holder. Wipe the surface of the blot holder with paper towel. Liquid must not come into contact with the film.
29. Place the film over the blots. For an unknown signal, start with a 10 second and 1 minute exposure.
Below is a modified sample buffer (SB) taken out of Current Protocols in Protein Science (see Electrophoresis chapter). It's called 6SB, but can be used between 1 and 2 strength (i.e. 2 strength, you would mix in proportion 2 parts sample + 1 part sample buffer) . Since it does not have 2-mercaptoethanol, it doesn't have to be used in the fume hood. Dithiothreitol (DTT) does not have as strong a smell.
6 SB
In a 15 ml conical tube, mix 7 ml 0.5 M Tris-Cl, pH 6.8
3 ml glycerol (Cut the tip off a 5 ml pipet and pipet slowly.)
0.02 ml 0.5 M EDTA
add 1.03 g SDS
0.93 g DTT
Put on the rotator until dissolved
Add 2 mg bromophenol blue
Mix until dissolved. Store in 0.5 ml aliquots at -70C.
The final concentrations of the components at 2 strength (recommended) is:
110 mM Tris-Cl pH 6.8
10% glycerol
0.33 mM EDTA
3 % SDS
182 mM DTT
0.07% bromophenol blue
Western Blot Transfer buffer:
2L of 10 Transfer buffer
60.6 g Tris base (Electrophoresis grade)
288 g glycine (Electrophoresis or tissue culture grade)
Dissolve in 2 L (final volume) MQ water
For 4 L of 1 Transfer buffer final concentration of 1
400 ml 10 Transfer buffer 25 mM Tris
2800 ml MQ water 192 mM Glycine
800 ml methanol 20% methanol
Ponceau Stain
Dissolve 0.5 g Ponceau S in 1.0 ml glacial acetic acid. Bring volume to 100 ml with MQ water.
Final solution is 0.5% Ponceau/1% acetic acid. Wrap bottle in foil to protect from light. Reuse the solution until staining is no longer as good.
10 TBS (Tris-buffered saline) 2L
Dissolve 48.4 g Tris base
160 g NaCl
in about 1600 ml MQ water.
Add conc. HCl (about 20 ml required) until pH is about 7.8.
Let sit for a few hours to cool to room temperature.
Adjust pH to 7.5 with 6 N HCl and bring volume to 2 L with MQ water.
For 1TBS, simply dilute 10-fold. Final concentrations are 20 mM Tris-Cl
136 mM NaCl
For TBS-T (1L), mix 100 ml 10TBS with 900 ml MQ water.
While stirring, add 1 ml Tween 20 (0.1% final).
Blocking Solution
For 2 blots, prepare 100 ml:
Weigh 5 g skim milk powder into a beaker.
Add 100 ml TBS-T and stir to suspend completely.
Western Blot Protocol
1. Cast Gel:
Assemble minigel apparatus( Be sure no leaking)
Make resolution gel (recipe) and mix
Make stacking gel (recipe) diwater—acrylamide/bis
Add 7.5ml of res gel to plates; Add diwater (to make sure it’s flat.)
(While waiting for resolution gel to harden (30min)
Clean up
Prepare running if needed (50ml 10X tris-sds500ml dwater)
Resolution gel should be hard now, pour off water, blot with filter paper
Finish stacking gel (temed, aps),shake gently. Add stacking gel until at top, not overflowing. ***Hurry and insert comb** **no air bubbles*****
Wait ~ 30min for gel to harden.
While waiting for stacking gel to harden:
2. Prepare samples:
1) Get ice, turn heat block on
2) Take samples out of freezerinto waterbath to thaw if needed then into ice bucket.
3) Label eppendorf tubes
4) Add ________ul of sample to tubes (= ug/ul)
5) Add _____________ to marker tube *make sure marker is at bottom of tube*
6) Add _____________ of 2X western blot dilution/sample buffer(Blue) to all
poke holes boil all tubes for 1-3 min.
3. Run gel:
Remove combs
Put both gels on plastic thing
Add running buffer (pour on side to prevent bubbles)
**eye level**
**To load samples, press 1st stop, place tip in bottom of tube, lift sample slowly, no bubbles!!!**
Load ______ul marker to lane 2; Load ______ul samples to wells
Assemble box Run ~1 hr at ~132v
4. Transfer electrophoresis:
Cut filter squares
Cut membrane film soak in plastic dish with methanol.
Soak foam pads in transfer buffer
***No bubbles in anything***
In box with transfer buffer, assemble sandwich
Gel is on big plate, so you know marker is on left side
Sandwich: 2 foam padssheet of filter paper place on gel 1st gel (no bubbles)membrane(no bubbles)filter paper2foam pads.
Place in box apperatus--clamp--fill with transfer buffer--no leaking—pour into side Run ~2hr at ~21v
Replace running buffer
Prepare plastic square boxes with 1X PBS
Disassemble box and place the membranes in PBS wash / remove PBS
Add 20ml milk buffer and rocker 20min.
Pour out
Add 10ml milk buffer with *1st AB __________________________ul in plates and rock 1hr.
(Could stop here and put in 4o over night.)
***SAVE AB***
Add 10ml milk bufferrock 10 min. / pour out
Repeat
Add 10ml milk buffer add 2nd AB ______________________________ul
Rock 30 min. / pour out milk
Add 10ml milk rock 10 min. / pour out
Repeat
Wash with 15ml plain buffer 20 min.
5. Develop film:
Take saran wrap and cut a piece for each membrane
In plastic sq boxes add detection reagent 3 ml. #1, and 3 ml. #2 ***mix***
Blot membrane on sides
Place membrane in detection reagent shake 1 min.
Blot membrane lightly on sides, place on saran wrap(in cassette) and fold over
Tape them down on the cassette / close
In darkroom ( red light) place film on membrane and close box for _1_min
Place film in autodev. Machine check film.
If good results, label film using marker, date, #, Ab used, protein used.
Western Blot Recipes
1. resolution Gel: 50ml 20ml 10ml
dH2O 24.2ml 9.48ml 4.78ml
1.5M Tris-HCL(pH8.8) 12.5ml 5.0ml 2.5ml
10% SDS 500ul 200ul 100ul
40% Acrylamide/Bis 12.5ml 5.1ml 2.5ml
TEMED 50ul 20ul 10ul
10% APS(Fresh) 250ul 200ul 100ul
2. Stacking Gel: 20ml 10ml 5ml
dH2O 12.66ml 6.28ml 3.1ml
0.5M Tris-HCL(pH6.8) 5ml 2.5ml 1.25ml
10% SDS 200ul 100ul 50ul
40% Acrylamide/Bis 1.95ml 1000ul 600ul
TEMED 40ul 20ul 10ul
10% APS 150ul 100ul 50ul
3. transfer Buffer: 1500ml
Tris Base( no HCL ) 4.54g
Glycine 21.7g
dH2O 500ml and mix
Methanol 300ml mix with 200ml dH2O
Bring to 1500ml with dH2O
4. milk buffer: 1000ml 500ml 250ml 100ml
Mix milk in beaker with 50ml dH2O 1st
Dry milk 50g 25g 12.5g 5g
Mix with rest in cylinder:
1M Tris-HCL(pH 7.5) 20ml 10ml 5ml 2ml
5M NaCl 27ml 13.5ml 6.7ml 2.68ml
Tween-20 (100%) 1ml 0.5ml 0.25ml 100ul
Bring up with dH2O to 1000ml 500ml 250ml 100ml
5. Plain buffer: 100ml 50ml 25ml
1M Tris-HCL(pH7.5) 2ml 1ml 500ul(.5ml)
5M NaCl 2.75ml 1.37ml 680ul(.68ml)
Tween-20(100%) 100ul 50ul 25ul
Bring up with dH2O to 100ml 50ml 25ml
6. 2X Dilution/Sample buffer 4ml
0.5M Tris-HCL(pH6.8) 1ml
Glycerol (100%) 0.8ml
10% SDS(Laryl Sulfate) 1.6ml
2-ME (-mercaptoethanol) 0.4ml
0.05% Bromophenol Blue(dye) 0.2ml
7. Stripping Buffer: see protocol
8. 0.5M Tris-HCL: 400ml
6.05g Tris base in 40ml dH2O
Adjust pH 6.8 with 1 N HCL
Add dH2O to 400ml total volume
9. 1.5M Tris-HCL: 500ml
91g Tris base in 300ml dH2O
Adjust pH 8.8 with 1N HCL
Add dH2O to 500ml total volume
10. 10X SDS Running Buffer/electrophoresis buffer:
Dissolve 10g SDS in beaker of dH2O (may have to heat)
Dissolve 30.2g Tris base
Dissolve 144g Glycine
Place all in cylinder add dH2O to 1000ml
To use, make 1X dilution: 50ml 10X add dH2O up to 500ml
100ml add dH2O up to 1000ml
11. Coomassie blue G-250 staining solution:
Acetic acid 20ml
dH2O 180ml
Coomassie blue G-250 0.05g
Mix 1 hr. store RT
12. Destaining buffer:
50 parts acetic acid
165 parts methanol
785 parts dH2O
1.用脱脂奶粉封闭的效果不一定比用BSA差,不过我们试过几种奶粉,有一些奶粉不太适合.推荐使用的奶粉:雀巢高钙奶粉,多力精低脂奶粉.一般国产的奶粉的颗粒比较大,不太容易溶解.
2.关于三名治:本人以前做时花很多时间在防止短路上,现在我做的时候根本没有剪齐,最主要的是赶气泡,这是关键的关键,其实转膜的原理是电流的单向流动,现在的好多公司的转膜装置的电极是一个平面,所以不用担心短路的.(不信的可以试试!!)
3.关于转膜时的温度:转膜时的温度比较重要,最好保持在10度以下,可以用冰浴(Pharmacia的转膜装置),也可以在转膜液中放入合适的事先准备的冰袋(-70度>3小时)(Bio-Rad),这样既可以降温又可以节约Buffer.
4.关于转膜电流和时间:一般转膜的电流在200mA-400mA之间时间为1小时,大片段的>50KD的可以选用350mA,小片段的可以用250mA,不过本人觉得上述电流均能很好的把大部分蛋白转过去.
5.关于反复标记和再标记:一张膜上可以同时标记很多抗体,但要注意,一般在两个目的蛋白相差在5KD以上的,最好先后两个抗体是不同的二抗.在两个目的片段相差小于5KD时,就需要经过处理,再标记了(Re-Blot),有些公司有再标记的现成试剂,不过比较贵,最简单的方法是:ddH2O洗5分钟,0.2MNaOH5分钟,再用ddH2O5分钟*3次,然后可以再标记了.要做多个抗体的膜\的选择,最好用PVDF或教坚固的,推荐用Amsham的Hybond-P,Hybond-Extra.本人最多在一张膜上做个10个不同的抗体.
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