Hello everyone,
so I have blocked my membrane in 5% BSA then incubated over night with primary antibody in TBST then secondary in 3% milk. when developing I saw the band I'm looking for. afterwords I wanted to reblot the membrane for a positive control so I incubated it overnight with the antibody in TBST , then the secondary in milk as well but this time I didn't get strong bands.. so now i have incubated the membrane once more in another primary antibody in order to have it as a positive control .
should I dilute the secondary in BSA in order to see a stronger band?
I don't know why my positive control did not give me clear bands as my previous blot.
I would appreciate any suggestions you may have.
Thank you
Hello Delilah,
It could be possible you need to stripp your membrane before reincubating. If you don't stripp and reincubate with the same antibodies which are already bound on the membrane, they will not bind and signal will be lost. So, you have to stripp your membrane in order to remove the secondary antibody from the antigen-antibody complex and uncover the epitopes once again. There are several stripping protocols, but, I'm used to employ this one for reblotting with the same antibodies:
Mild stripping
Stripping Buffer (1 liter)
15 g glycine
1 g SDS
10 ml Tween20
Adjust pH to 2.2
Bring volume up to 1 L with ultrapure water.
Membrane incubation with stripping buffer. Use a volume that will cover the membrane. Incubate at room temperature for 5-10 minutes.
Discard buffer.
5-10 minutes fresh stripping buffer.
Discard buffer.
10 minutes TBS
10 minutes TBS
5 minutes TBST
5 minutes TBST
Ready for blocking stage.
I hope you will can resolve your problem.
Hello Delilah,
It could be possible you need to stripp your membrane before reincubating. If you don't stripp and reincubate with the same antibodies which are already bound on the membrane, they will not bind and signal will be lost. So, you have to stripp your membrane in order to remove the secondary antibody from the antigen-antibody complex and uncover the epitopes once again. There are several stripping protocols, but, I'm used to employ this one for reblotting with the same antibodies:
Mild stripping
Stripping Buffer (1 liter)
15 g glycine
1 g SDS
10 ml Tween20
Adjust pH to 2.2
Bring volume up to 1 L with ultrapure water.
Membrane incubation with stripping buffer. Use a volume that will cover the membrane. Incubate at room temperature for 5-10 minutes.
Discard buffer.
5-10 minutes fresh stripping buffer.
Discard buffer.
10 minutes TBS
10 minutes TBS
5 minutes TBST
5 minutes TBST
Ready for blocking stage.
I hope you will can resolve your problem.
Hi Delilah,
you means you want to recycle the membrane?
if you want reblot the membrane, you could recycle your membrane quickly and wash it three times by TBST 10min per, then following your WB process from incubating BSA or milk. and then reincubate with your first AB and second AB.
Some advice，
1 the band of reincubating first AB is stronger than your first AB, if the two protein molecular weight are simlar.
2 maybe you could try incubate second AB without milk, and just TBST.
I simply incubate the membrane with 0.5N NaOH for 5-10 min and then wash 2x with TBST.
The membrane is now ready for probing another primary antibody directly without blocking/re-activating the membrane.
Hi Delihah,
there is some missing information about your WB. Firstly, which type of membrane did you use? Nitrocellulose or PVDF? If it is PVDF, which is the pore size of the membrane (0,2 um or 0,45um)? Because if your positive control has a very low weight (
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