Storing western blot after primary

[sodium_azide]

Hi All -

I am responsible for a summer student who also has classes; as such I have found it difficult to get her into lab for a long period of time to get stuff done (not her fault). Anyway, I am teaching her Westerns, but unfortunately she cannot come in till tomorrow. She put a blot in primary overnight last night, and it is ready today. Of course, I could always put it in secondary and image it for her, but this teaches her nothing! So is there anyway I can store the blot for another night so that she can put it in secondary and image tomorrow?

Cheers,
Robert

[mdfenko]

you could put it into blocking solution.

[relaxin]

Yes, I have stored a blot in blocking buffer at 4 C over one month, while waiting for the shipment of an antibody. It worked.

Another time, the blot dried out while on storage in the refrigerator. I reactivated the PVDF blot with methanol, washed with TBST, reblocked, and incubated with a different antibody. It still worked.

[sodium_azide]

Thanks for the replies guys. I ended up putting it in TBS over last night, without any washing. But if I ever need to do this again I will use a blocking buffer.


Cheers!

[gsmejkal]

I routinely dry Western blots.

Back in the day we used radiolabled proteins, we quantified the retention of protein antigens on nitrocellulose and PVDF membranes. Drying the membrane down prior to immunoprocesses actually "fixes" some proteins.

On the other hand, storing the membranes in blocking reagent for a month is risky. More so for nitrocellulose, where the high protein concentration of the blocking solution drives the loss of specific protein (e.g. your antigen) from the membrane. (Millipore has a Technical Bulletin to this effect.)

I have successfully dried down membranes after incubation with primary antibody and after washing away unbound. Later, rehydrated the membrane and proceeded with secondary antibody with no deleterious effect.

[seankim]

Hi All -

I am responsible for a summer student who also has classes; as such I have found it difficult to get her into lab for a long period of time to get stuff done (not her fault). Anyway, I am teaching her Westerns, but unfortunately she cannot come in till tomorrow. She put a blot in primary overnight last night, and it is ready today. Of course, I could always put it in secondary and image it for her, but this teaches her nothing! So is there anyway I can store the blot for another night so that she can put it in secondary and image tomorrow?

Cheers,
Robert

just keep it in primary ab solution at 4c. and have her wash it and do the secondary/wash/imaging tomorrow. if she can't, then leave it in secondary overnight and wash and image the next day.