3 replies to this topic

[bluelady09]

Has anybody ever used fish gelatin for blocking?

Also if I forgot to briefly rinse my membrane after blocking and I just directly added my diluted Ab will this affect the results significantly?

I blocked the membrane with the same blocking agent that I dilute my Ab in. So i thought this should be ok.
Has anybody done the same mistake as I did? and still got good results?

=(

[rkay447]

I've tried the cold fish skin gelatin for blocking and saw nothing better than the blocking with 5% BSA so I decided to use the BSA.  The gelatin smells horrible and I hate working with thick, syrupy solutions.  However, I just realized you are talking about a membrane.  I've only used the FSG for blocking coverslips for IF.  No worries on the rinse after the block.  Most of the time I don't rinse before tossing on the antibody and have never had a problem.

[stardust]

Hi

I have used it and was not very happy with it...tried 0,5 - 5 % fish gelatine in TBST...BSA 5 % in TBST was much better

Stardust

rkay447, on Feb 7 2009, 04:05 PM, said:

I've tried the cold fish skin gelatin for blocking and saw nothing better than the blocking with 5% BSA so I decided to use the BSA.  The gelatin smells horrible and I hate working with thick, syrupy solutions.  However, I just realized you are talking about a membrane.  I've only used the FSG for blocking coverslips for IF.  No worries on the rinse after the block.  Most of the time I don't rinse before tossing on the antibody and have never had a problem.

[unity]

bluelady09, on Feb 7 2009, 03:09 AM, said:

Also if I forgot to briefly rinse my membrane after blocking and I just directly added my diluted Ab will this affect the results significantly?

I never wash the membrane between blocking and incubating with Ab, only I block it with 5% milk, not with gelatin.

bluelady09, on Feb 7 2009, 03:09 AM, said:

I blocked the membrane with the same blocking agent that I dilute my Ab in. So i thought this should be ok?

I also do it every time, the only difference is that the milk I use for blocking is 5% and for Ab is 1%.

I guess, when you use the same solution for blocking and for dilution of your Ab, it doesn't matter if you wash it beetwen or not.