PAGE storage and tubulin antibodies

[tyronegenade]

Hello,

1. Once the PAGE is prepared, how long can it be stored and under what conditions? For instance, if I prepare the gel on Friday afternoon, can I put the set gel in tank buffer and then store it in the fridge till Monday? Or would simply wrapping the gels in mQ H2O soaked paper towels work just as well?

2. I'm having trouble with our Santa Cruz alpha-tubulin antibody (SC-8035). It doesn't work well---its temperamental, sometimes it works and other times not and we don't know why. Anyone else having these problems? Found a solution? Alternatively, can one suggest another anti-house-keeping antibody which can be used as a loading control but also has multispecies activity. Ideally, I'm looking for something active against rat, goldfish and zebrafish. An alternative alpha-tubulin antibody would be most welcome.

p38 has been suggested and this would be great as my target proteins are all much larger than 38kDa but does it have multispecies activity?

Thanks

[seena]

Hello tyronegenade
We usually prepare the gel in advance and store it in polythene bags with enough transfer buffer to keep the gel from drying out.It works fine for us.

[tyronegenade]

Thanks for the reply Seena.

[quest]

I have been using the Alpha-tubulin antibody from Sigma with catalog No # T5168.
It works perfectly well in all my blots. The protocol that I follow is -
Blocking for 45mins, 3x5min washes in washing buffer, Primary Ab incubation for 45 mins, 3x5min washes in washing buffer, seconday Ab incubation 45 mins and final 3x5min washes in washing buffer, 5 min wash in PBS and go for chemiluminescent detection. The washing buffer is 0.05%PBS-T and the dilution for blocking and Primary, secondary dilutions is 3%Milk in 0.05%PBS-T. Everything should be done at room temperature. Hope you will have good results.

[tyronegenade]

Thanks for the reply Quest. I will try the tubulin antibody again next week and see if PBS makes the difference.

Thanks