Dear all,
I performed Western blot on plant material by using specific antibody raised against rabbit. When I try to detect the proteins, I see background from the nitrocellulose membrane (see annex). Briefly, I block the sample with PBST 0.1% + 5% milk for 1h at 4 degrees; I Ab 1:5000 dilution, II Ab 1:2000 dilution; incubation time both 1h at 4 degrees, 2x5 minutes washes in between. After that, I use 1ml of detection mix, EZ-ECL solution A and B, by wrapping the membrane on nylon wrap, then I drain off the excess and analyse with SynGene software. On the picture uploaded, I also see stripes of the wrap onto the membrane, and, of course, background.
Does anyone have any suggestion for this inconvenience?
Many thanks.
Looking forward to hearing from you,
Chiara
Hi Chiara,
I feel its a blocking issue (membrane is not blocked enough). Why not try alternate blocking agents such as Tween alone, soy protein or non-fat skim milk.
Good luck,
Sabarinath
Hi Chiara,
I feel its a blocking issue (membrane is not blocked enough). Why not try alternate blocking agents such as Tween alone, soy protein or non-fat skim milk.
Good luck,
Sabarinath
Blocking and washing issue. After primary antibody incubation you needs to wash properly.
Try to block the nitrocellulose membrane with 3% NP-40 in PBS/ 30 min and then, wash the membrane 3 x 5 min with PBS/ shaking.
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