1. Optional: "Renature" gel- this is thought to permit some refolding of proteins and may be important in finding epitope recognition of monoclonal antibodies. However its major advantage is probably that it removes the SDS from the gel which impedes transfer.
Soak for 30 min- 2 hrs in: 10 mM Tris pH 6.8 0.1 M DTT 4 M Urea 50 mM NaCl 2. Transfer 2-3 hrs- O/N at 350mA 3. Block in TBS containing 0.05- 0.1% Tween for 15 mins to O/N OR + 5% non-fat dryed milk for ECL at RT for 2hrs 4. Stain with Ponceau S for 5 mins 5. Dilute antibodies in blocking buffer. 1/500 for 125I blots for 2hrs RT 1/-5,000 for flag & myc and 1/3000 for 5172 for ECL O/N at 4C 6. 3 X 10 min washes in TBS-Tween. 7. Incubate in Rabbit anti-mouse 1-2 hr at RT (dilute 1/500) 8. 3 X 10 min washes in TBS-Tween.
9. Secondary antibody 1-2 hr at RT
(Use NEX-L- low specific activity 125I-protein A -diluted 2 x 105cpm/ml
(1uCi = 106 cpm)
dilute Goat anti mouse HRP-ab 1/4,000 for ECL
Add 1M NaCl and 0.5% Triton X-100 to the middle wash for 30 mins to 1 hr if background is a problem. ECL Incubate 1min @ RT in mix (1:1) of the two ECL solutions Dry out the excess liquid on Whatman paper Wrap the membrane in Saran wrap 10X Tranfer Buffer 30.27g/L Tris 8.8 -25mM 144.1g/L Glycine -192 mM ECL reagent: homegrown version Advantages: almost no cost; stable/reproducible, stored in frozen aliquots To 10 mls of 100 mM Tris pH 8.5 (RT), add 50 ?ls of luminol (warm to redissolve) 22 ?ls of coumaric acid (warm to redissolve) 3 ?ls of H2O2 (fresh < 6 months) Pour onto blot for 1 minute and process as normal (10 mls enough for 100 cm2) Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266 mgs in 6 mls DMSO; store frozen in 60 ?ls aliquots. Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in 2.5 mls DMSO; store frozen in 25 ?l aliquots. Stripping blots: Most antibodies should be removed by heat and SDS: 100 mls ?-ME 100 mM 0.71 mls 14.1 M stock from bottle SDS 2% 20 mls 10% stock 62.5 mM Tris, pH 6.8 12.5 mls SDS-PAGE upper gel buffer Wash blot well with aq, including warm water, add stripping solution warmed to 50oC in closed container, incubate 30 mins, decant with large volume of running water, wash again with aq, and put into Blotto to reblot. Would recommend putting back into ECL to check efficiency of strip, and repeating the secondary control to check for background. Note foreground goes down with every strip, and background comes up.
To strip membrane after immunoblot - Heat Detergent-Manue
Stripping buffer: 100 mM ?-mercapto-ethanol, 2% SDS, 62.5 mM Tris-Cl pH 6.8
- ?-mercaptoethanol 176 μl 342 μl
- 20% SDS 2.5 ml 5 ml
- Tris-Cl pH 6.7 1.56 ml 3.125 ml
- to H2O 25 ml 50 ml
Incubate and agitate the membrane in stripping buffer for 30 min. at 50°C
Wash 6 X for 5 min. minimum each in 1X PBS - 0.1% Tween
Optional:
Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent
Expose the film for 1 min. to 1 hr to make sure that the original signal is removed
Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween
The membrane is ready to reuse
Start at the blocking step
To strip membrane after immunoblot - Low pH-Manue
Stripping buffer: 25 mM glycine-HCl, pH 2, 1% SDS
- Glycine 1.876 g
- 20% SDS 50 ml
- to H2O 1 L
Incubate and agitate the membrane in stripping buffer for 30 min. at room temperature
Wash 3 X for 10 min. each in 1X PBS - 0.1% Tween
Optional:
Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent
Expose the film for 1 min. to 1 hr to make sure that the original signal is removed
Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween
The membrane is ready to reuse
Start at the blocking step
Membrane: Protran (Schleicher and Schuell)
Rq: a PVDF membrane enables to sequence direcly from the membrane
Red ponceau S
Prepare a 2% (2 mg/ml) stock solution of Ponceau S dye in 30% (wt/v) trichloroacetic acid
Dilute the stock 10-fold in 1% acetic acid (final concentration of Ponceau S is 0.2%)
1X PBS - 0.1% Tween
- 5X PBS 200 ml
- Tween 20 1 ml
- to H2O 1 L
5% non-fat dry milk in 1X PBS - 0.1% Tween
- Non-fat dry milk 5 g
- to 1X PBS - 0.1% Tween 100 ml
Rq: this solution can be replaced by 3% BSA - 1X PBS - 0.2% Tween - sodiumazide (see Rodger)
Paint Pen Polymark: Natural Glow No. PM501, Dimensional Fabric and Graft
近日，MarketsandMarkets咨詢公司發布了全球westernblotting市場的分析報告。據MarketsandMarkets預測，全球westernblotting的市場規模將從201......
近日，MarketsandMarkets咨詢公司發布了全球westernblotting市場的分析報告。據MarketsandMarkets預測，全球westernblotting的市場規模將從201......
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