5 replies to this topic

[jasmina]

I dont success to see  a signal of my low molecular weight protein (16kda), I did 15% SDS-PAGE,and I blocked membrane in 5%milk, primary (monoclonal) and secondary incubations diluted in 5% milk as well.
Some people suggest to me to try BSA instead of milk .
Actually,  I incubated again  the same filter  with antibody diluted in milk overnight 4°C.
I wonder if I can do secondary antibody diluted in BSA?? or I need to keep it in milk?
thanks

[mdfenko]

yes, you can.

[acquire]

luciana what happened to yor blot.i want to know the result.iam going to work on 17kd protein blotting in future.

[jasmina]

Hi,
after blocking again the filter with BSA 5% and antibody diluted in BSA. I got a signal with background.. But, I noticed that 15% isnt really necessary. I can keep 10% becos It seems that this protein has a trimer at 55kda. Iwasnt able to see 16kda!

[acquire]

washes do for 10 mins each 3times after blocking,primary ab incubation and secondary ab incubation,this will reduce back ground properly this happend to me. in your case trimer your telling but  is your gel ,SDS reducing gel right? then how come trimer? have u heated sample for 5mins boiling before loading.?

Edited by acquire, 08 December 2011 - 04:05 PM.

[mdfenko]

acquire, on 08 December 2011 - 04:05 PM, said:

washes do for 10 mins each 3times after blocking,primary ab incubation and secondary ab incubation,this will reduce back ground properly this happend to me. in your case trimer your telling but  is your gel ,SDS reducing gel right? then how come trimer? have u heated sample for 5mins boiling before loading.?

i wouldn't worry about washing after blocking, especially if you prepare your primary antibody in the blocking buffer.