Remove blocking reagent without stripping? - (May/11/2006 )
Hi,
I have blocked my membrane with 5% non fat dry milk and detected via ECL.
Now I want to blot for another protein with antibody that develops high bakcground with dairy products.
I wanted to block with 5% donkey serum.
To do so I have to remove the NFDM bound to the membrane before.
Do I have to stripp it (My stripping buffer recipe: 62mM Tris pH 6,7; 2%SDS, 100mM BME) before?!?
Wanted to avoid this because of protein loss on membran...
if you strip the blocking agent then you will also remove your protein. your not just releasing antibodies when you remove your block you are removing all protein from the membrane. you will need to rerun your gel and transfer and block with the donkey serum.
what are you trying to detect? serum may contain too many different proteins for your purpose. you may want to block with bsa instead.
what are you trying to detect? serum may contain too many different proteins for your purpose. you may want to block with bsa instead.
You mean stripping always removes the top layer of proteins from the membran?
And this is the priciple of stripping? Always thought it would split the bond between protein and first antibody and the loss of protein was an inevitable side effect...
what are you trying to detect? serum may contain too many different proteins for your purpose. you may want to block with bsa instead.
want to do a anti-myoglobin blot alkaline phosphatase based
got the protocol from somebody and she said it would work well apart from a high background problem
told me in no case to use nfdm or bsa
hi kylvalda ! i totaly agree with mdfenko. stipping removes upper layer of proteins . but you intend to remove blocking agent which is attached to the membrane and this requires harsh conditions. that means you also loose your proteins under study bound to the membrane.
you have to start afresh. this time use a blocking reagent that that is compatible with both the antibodies/ proteins under study. may be "gelatin"..............
all the best
you have to start afresh. this time use a blocking reagent that that is compatible with both the antibodies/ proteins under study. may be "gelatin"..............
all the best
How do you block with "gelatine"?
Do you have a recipe or protocol?!?
hi kylvalda ! using gelatin is just like any other blocking reagent just prepare the required percentage of gelatin (1-2 %) in PBS /TBS, dissolve completely and use as usual.
all the best