I activated my membrane twice in total and hybridized it with two different antibodies. The first antibody was the specific antibody I was looking for and the second one was GAPDH. I then activated my membrane again, washed it in PBS and incubated my membrane in Restore Plus Western Blot stripping buffer (Thermo Fischer) for 20 min at room temperature. I removed the stripping buffer, washed my membrane and blocked it with 1% milk for 60 min and added my primary antibody. The next day I washed, added my secondary and so on. However, it seems all my previous hybridizations are still present and the stripping did not remove any of them. I did the stripping buffer incubation for 20 minutes rather than 5 to 10 minutes that they recommended to make sure it works!
Anyone has a similar experience and could it be that because I hybridize my membrane more than once, the stripping buffer did not work.
Appreciate your thoughts.
A PVDF membrane is highly recommended to minimize loss of sample protein. Note also that colorimetric/chromogenic detection reagents will leave a permanent visible stain on the membrane that can interfere with subsequent detection of targets of similar molecular weights. Chemiluminescent reagents such as ECL are recommended as they will not leave a stain and are more sensitive than colorimetric reagents.
From abcam.
Hi Vahid,
If the second antibody is very good, you can opt for a slightly harsher stripping buffer that you can prepare in house, 0.5M Tris.HCl pH 6.8, 100 mM β-mercaptoethanol and 2% SDS and warm the buffer to about 50°C before incubation. The downside here is that you will lose some of your proteins on the membrane. I am attaching a alink to the protocol for reprobing by Abcam. Their harsh buffer seems little different from what I use but the process is the same. Only, they recommend incubating the membrane at 50°C for 45 mins, which I think is overkill. 30 mins at RT on a rocker seems to do the job fine.
And if you want to be more cautious, before reprobing with the next antibody, you can add some secondary antibody and develop to confirm if the first antibody is completly gone without any background.
https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol
I understood that the PVDF is activated with methanol before transfer to reduce its hydrophobicity. This allows the aqueous transfer buffer to soak through and aids the proteins that are in phase with this buffer to bind to the membrane. Activating the membrane once before transfer should be sufficient becasue I never had any problems otherwise, even for blots with mutiple reprobes. The methanol might have denatured and fixed some of the antibodies onto your membrane. It kinda explains why they were hard to strip away. I am not sure about this assumption but it is the one thing that stands out to me.
Thank you for your answers.
I use ECL Junjie Shao
Praveesh Valissery I activate my PVDF membranes with ethanol before the transfer and each time I am going to use the membrane for any antibody. So I will not activate my membrane other than before transfer and see what happens.
Drying out your membrane will denature the antibodies and potentially cause background on your blot. So keep membrane wet at all time post Ab addition.
After stripping of membrane carry out the ECL on the membrane only and see if you get background on film. This will save you time in stripping and re-probing to finally find out there is a problem
To just get rid of secondary Ab a good wash with PBS (0.05 - 1% Tween) and o/n with changes did the trick for me.
If GAPDH is a different size to your protein of interest cut the membrane prior to probing and incubate membranes with appropriate Ab. Therefore no need to strip.
Usually, the previous bands will remain present, even after stripping the membrane. But the point is that they are much weaker and accordingly you can discriminate it from the new sharp bands. You can also probably omit them by reducing exposure time (the time placing a film on the membrane). Good luck
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