Dear All,
Is it ok to leave membrane incubating with primary antibody (dilution with blocking buffer) at cold room (4°C) for a few day?
I usually incubate primary overnight, but sometime it's on Friday and I prefer continue on next Monday. If I incubate the membrane with primary antibody for weekend, will the membrane has more background or need more wash? even incubating antibody dilluted in blocking buffer (5% milk or bsa)?
Thank you
Cheerios,
It depends on the antibody really. Our goat peptide antibodies have been affinity purified and may cause background when incubated for too long at too high concentration. We always recommend 1h incubation at room temp. It also depends on the titre of the antibody. Strong titre antibodies work best at a short incubation and weak tire antibodies require longer incubation (at higher concentration).
Leaving the blot in the blocking buffer overnight or over the weekend at 4C does not hurt.
I don't for see any big problems but you should check results with previous experiments.
Its always better to wash thoroughly when incubating longer.
i should use bsa but milk wont be a problem aswell.
It depends on the antibody really. Our goat peptide antibodies have been affinity purified and may cause background when incubated for too long at too high concentration. We always recommend 1h incubation at room temp. It also depends on the titre of the antibody. Strong titre antibodies work best at a short incubation and weak tire antibodies require longer incubation (at higher concentration).
Leaving the blot in the blocking buffer overnight or over the weekend at 4C does not hurt.
Always primary antibody to use maximum overnight or 24 hrs (4°C) . Membrane store in PBS or TBST buffer 4°C weekend or 2 to 4 days fine.After block 5%milk and continue the further step.
I've done this often and it does not result in excess background with the antibodies I've been working with. One caution is that you need to cover the tray tightly to prevent evaporation and make sure your incubation volume is sufficient. Also, if you're incubating in milk, make sure it's fresh, or it may go bad over the weekend.
I have left primary antibody for 2 days and it was fine . Just wash the membrane thoroughly before adding the secondary
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Clarity of solutions and dishes is question in the case of longer incubation. Only for first ab, i should suggest some azide in the incubation buffer.And the above considerations.
Hi....you def can do weekend in PBST but if you generally incubate primary overnight then that doesnt save you time really at all...i am sure you can try primary over the weekend and it should in principle work (if with a higher background but if antibody is clean it wouldnt matter)....azide wouldnt help if your milk goes bad during the incubation....i doubt thats a problem though for 2 days...
good luck,
I agree with above answer, but I felt the milk quality influence alot. If you bought milk powder from local store like me I wont let it incubate over the weekend in blocking or primary. Blocking too long slightly reduces primary antibody binding ability and it become significant when u use some weak antibodies. Incubate primary antibody over the weekend (4C) works, sometime I noticed more background and I just give it couple more washes at 4C and it helps clear things up.
I don't know whether this would be of any use right now..
I usually dry my PVDF membranes and reactivate in methanol briefly. You may be able to dry the membranes on Friday and reactivate it on Monday (option 1).
If you're worried about the background, you may be able to block the membranes O/N at 4C before incubating with the primary over the weekend (option 2).
I'd rather take option 1 since reproducibility could be an issue when you vary the time of primary incubation.
I try to avoid incubating over the weekend. I would just wash my blot and dry over the weekend, then rehydrate on Monday and begin the process then. I have blocked then washed, dried and hit with antibody the next day or on Monday with no issues. I've learned that you will get a lot of background if using 5%milk and it also weakens the antibody. I have switched from milk to BSA for my blocking.
you can block in 3% milk , avoid incubation more than one day, also you check your protein concentration
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