How long can a protein last on membrane? - (Sep/07/2006 )
I'm gonna to detect phosphrylated Akt and total Akt in Western Blot. Because protein amount is limited. I can only run one lane per sample. So I need to probe with phospho Akt o/n, then re-probe with b-tubulin o/n, then strip and probe with total Akt o/n, then b-tubulin o/n. So I need at least 5 days to do the experiment. But I'm not sure whether protein levels of total and phospho Akt are still comparable if detecting them in different days. Is protein gonna degrade over a number of days? And how should I keep proteins still on my membrane the next week if I can't get access to the developing machine in weekend?
I store my dry PVDF membranes at 4oC for months and the proteins are still there
I store my dry PVDF membranes at 4°C too. and proteins stay on it!
If you although want to be sure you didn't loose proteins, you could blot for phospho-Akt, then strip and then blot for Akt. So blotting for tubulin at the end (since it's only a loading control...).
Why do you want to blot 2 times for tubulin?!
I think there's no need to blot twice for tubulin. This is a very abundant protein and I don't think it will give you an indication of how much you lost during the stripping procedure, if this is the reason why you want to do it twice...
most of the stress to a blot appears to be stripping; even some harsh strip protocols do not cost appreciable amounts of protein; short time storage (2-3 days) can be done in PBS/TBS, cooling might not be necessary, long-time storage after stripping and washing in water, drying and stored at at least -20°C in evacuated sealed bags
Is is possible to cut the membrane in half horizontally so that b-tubulin would be on one half and Akt on the other half? Or are they too close in MW? This could save a stripping step if it's possible.
Thanks for your replies, I'll do b-tubulin once as most of you suggested.
No, b-tubulin is 55KD. Akt is 60KD. I hope they can be separated on a 12% gel. I can't expect they are far apart enough to be cut in middle.
Actually, they're too close to be separate by cutting the membrane. But there's no need of this.
We often work with p-Akt and Akt and others phospho-proteins in Western Blot and we proceed always like this: first, p-prot; second, prot; third, loading control.
we are also interested in proteins in the range of 50-60 kDa, so tubulin is not a good choice for us; we use instead beat-actin as homekeeping protein control, it is of 40 kDa; proposal of cutting membrane is more easier to perform in this case