I've done Western Blotting using total Akt, pAkt ser 473 and pAkt thr 308. For total Akt and pAkt ser 473, I mostly see a very clear and sharp band. But there are so many non-specific bands for pAkt thr 308. All of the conditions are the same. I use 5% non-fat milk to block and 5% BSA to dilute the primary antibody.
Thank you!
Thanks! We use the cell signaling antibody. I do run separate gels. I've never stripped the membrane. I can see that they are at similar sizes but there are just too many non-specific bands for thr 308.
Hi Jingyi,
Is there any reason you can't do an IP to purify the specific protein, then do the WB using your thr308 probe?
Also, can I ask what your washing protocol is between antibodies?
Hi Gary,
That's a possible option, but it's a bit costy for this type of experiment. We use TBST (0.1% tween 20) to wash the membrane.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies