If I have run and developed a western blot PVDF membrane, but wish to redevelop it with different antibodies, how do I strip the membrane of primary and secondary antibodies? Is it completely necessary to strip the membrane? Do I have to re-block the membrane once it is stripped?
Thank you.
Hi David
One can definitely reprobe a membrane but the procedure depends on number of parameters.
1. If the protein B is running at different size then the previous proteins (A) then you can simply give a try without any stripping.
2. If the protein B is running at the same size as previous ones: In this case there are two possibilities:
a:. If the primary antibodies are raised in different species then do not strip. Simply add around 0.1/0.5% of sodium azide to your primary antibody solution while incubation. After incubation, wash at least 30 min in TBST (with 4-5 changes) to get rid of all the sodium azide. Now add your secondary and proceed as usual.
This can be used for the case 1 as well if one faces a problem due to strong signal from the previous antibody (provided primary antibodies are raised in different species).
Note: Sodium azide destroys the HRPO activity of the secondary antibody from the previous blot so it does not react with reagents during subsequent reactions.
b. If the primary antibodies are from the same species: here stripping is necessary. Now you can use a mild or a stringent stripping buffer depending on sensitivity of the antibodies. We incubate membranes in 0.2 M NaOH for 10 minutes for mild stripping. For stringent stripping we use the following protocol:
Article A solution for stripping antibodies from polyvinylidene fluo...
After stripping you have to repeat washing and Blocking.
Thanks
Cheers
Prafull
I have an experience of this, what I followed is as under:
After taking the images of bands,
First wash the PVDF membrane (with TBST) over 150-200 min (slow shaker)
Then wash with again for 30 min moderate shaker (twice).
Then follow the blockage procedure, (in our lab we use 5% milk for 60 min at room temp at slow shaker)
Then wash again with TBST (3 times)
then incubate for primary antibody over (10-14 hours at 4 degree).... and so on.....
We usually do this for the proteins that have very close molecular weight. Usually, the protein with less/weak/thin band visibility (relative to the other protein on same membrane to be assessed in 2nd turn) are analysed first, and the the protein with (relatively) more expression are assessed in 2nd turn.
Here the trick is, re-using of the membrane need little more time for washing, blocking and primary antibody incubation.
Blot stripping can be mild or harsh depending on the need. Mild stripping generally involves moderate rocking of the blot in mild stripping buffer; harsh stripping involves incubation of the blot in harsh stripping buffer for 5 minutes.
Following this, the blot is to be washed with tap water for 10-15 minutes and later with TBST for 5-10 minutes.
Post washing, the blot has to be blocked and then incubated with the appropriate primary antibody.
Stripping buffer composition generally includes Tris-HCl 6.8, SDS, Beta Mercaptoethanol and water.
I think the abcam protocol shared by Xin Pin has the details of the composition.
Hi David
One can definitely reprobe a membrane but the procedure depends on number of parameters.
1. If the protein B is running at different size then the previous proteins (A) then you can simply give a try without any stripping.
2. If the protein B is running at the same size as previous ones: In this case there are two possibilities:
a:. If the primary antibodies are raised in different species then do not strip. Simply add around 0.1/0.5% of sodium azide to your primary antibody solution while incubation. After incubation, wash at least 30 min in TBST (with 4-5 changes) to get rid of all the sodium azide. Now add your secondary and proceed as usual.
This can be used for the case 1 as well if one faces a problem due to strong signal from the previous antibody (provided primary antibodies are raised in different species).
Note: Sodium azide destroys the HRPO activity of the secondary antibody from the previous blot so it does not react with reagents during subsequent reactions.
b. If the primary antibodies are from the same species: here stripping is necessary. Now you can use a mild or a stringent stripping buffer depending on sensitivity of the antibodies. We incubate membranes in 0.2 M NaOH for 10 minutes for mild stripping. For stringent stripping we use the following protocol:
Article A solution for stripping antibodies from polyvinylidene fluo...
After stripping you have to repeat washing and Blocking.
Thanks
Cheers
Prafull
- Badges
- Science method