I stipped and reprobed my PVDF membrane 4 times using 3 different antibodies (bax, bcl-2 and p53) and loading control (GAPDH), then I stripped and reprobed the same membrane with GAPDH again (as confirmation), but the intensity reduced about three folds compared to the previous. Did the “stripping and reprobing” cause loss of targeted protein?
This might be a very likely cause. A little trick that may allow you to reuse your membrane many times without substantial loss of bound protein is to dry it completely after transfer. Before probing with first antibody just soak it briefly in methanol and wash it with TBST. Then bound proteins will be a nearly permanent record that can be subjected to multiple rounds of stripping and reprobing without loosing too much. On the side of this protein loss may also be related to the mode of stripping. Just check Abcam web page for different stripping alternatives. Hope this helps
This might be a very likely cause. A little trick that may allow you to reuse your membrane many times without substantial loss of bound protein is to dry it completely after transfer. Before probing with first antibody just soak it briefly in methanol and wash it with TBST. Then bound proteins will be a nearly permanent record that can be subjected to multiple rounds of stripping and reprobing without loosing too much. On the side of this protein loss may also be related to the mode of stripping. Just check Abcam web page for different stripping alternatives. Hope this helps
Which method do you use to strip your blot? I would suggest reducing the treatment duration and try it again. if the protein sizes are different then you can try cutting your blot and do all westerns simultaneously. in my case i could reuse my blots till 3 stripping treatments. then it would go away. As a good measure detect GAPDH the last as its way abundant and it's still there after stripping treatments
Thanks George.
Dear Abbas,
I used the following for my stripping buffer:
total volume=25 ml
1.56 ml 1M Tris ph6.8
5 ml 10% SDS
0.175 B-ME
18 ml dH2O
I added to the membrane and rock for 30 min at 50 degrees celcius. then, wash at least 3x with TBST (30 min each time). after washed are completed I block my membrane again as usual and reprobe the next antibody.
Dear Amran
I think this protocol is for harsh stripping. You try once with mild stripping protocol (Abcam). Definitely it will help you.
Hi Khairul Arman, When I used GAPDH as loading control, it did not work. Can you please share protocol of yours on using loading control ? Thanks
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