1X Stripping buffer (g/L):15.0 Glycine, 1.0 SDS, 1% Tween 20, pH 2.2
-membrane was agitated two times with stripping buffer 5-10 min. each.
-The membrane was then washed two times with PBS 10 min. each followed by two times washing with TBST 5 min. each.
-The immunodetection step in western blotting protocol was repeated
this protocol worked perfect with me in case of detection of phosphorylated protein and their total
10 ml SDS 10%, 6.25 ml Tris HCl pH6.7, 39.75 ml H2O, mix and them add 390 ul 2-mercaptoethanol, 30 min 50ªC
I have gotten pretty good results with a 0.5 M NaOH (from solution , not powder). Incubate the blot for 10 - 20 min at room temp in the stripping buffer, wash 2x in H2O and you are ready for blocking.
please note that difference in recipes is according to how harsh conditions you want.. according to Abcam protocol and trials start with the mild buffer then go to more harsh conditions if mild conditions are not working http://www.abcam.com/ps/pdf/protocols/Stripping%20for%20reprobing.pdf
Have you tried Western Reprobe from G Biosciences? This is a good mild stripping solution with 2-4 hour incubation. Does not work in every case (especially if signal was very high) but can be very helpful.
What kind of membrane do you have? If its PVDF, did you pre-incubate the membrane in methanol?
2% SDS, 10mM Tris-Hcl pH 6,8 2 hours a room temperature and then wash three time with water for 20 minutes
Thank you...Can i use these recipe for Nitrocellulose membranes?
Yes, you can try.
there are two ways of stripping.
1.stripping by heat and detergent. stripping solution: 100 mM 2-bME, 2% (w/v) SDS, 62.5 mM Tris Hcl, ph6.7 Incubation - 30 minutes at 50C in stripping solution. Then 10 min in PBS. Then block the membrane. Repeat initial immunodetection protocol.
2. stripping by low pH.
stripping solution -25mM glycine-Hcl, ph2, 1% (w/v) SDS.
Incubation 30 min in stripping solution with agitation at room temp. Then 10 min in PBS. Repeat 2 times. Proceed to block the membrane.
Good luck.
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