Hi,
I have a question regarding western blot. I performed total protein statin after trasfering membrane. The result was totally fine before (Figure 1). However, there were strange bubble-like spots over the membrane every time since last month (Figure 2). I have tried more than 10 times and switched the gel, PVDF membrane, buffers and the results were the same. I transferred the membrane under 200mA for 3 hours. Every one in our lab use the same transfer condition and share the tank for transfer. Their results were totally normal. I was wondering what is the problem?
Hi, it looks like u spilled a proteolytic compound, or maybe some sort of acid/base? Protein is clearly no longer there is the second picture.
Vatamanu Sergiu-Marian Hi, it was my guess in the begining. But I have chaged several PVDF membrane from different boxes and the results were the same.
Hi Chien-SWei,
difficult problem. I first thought about air bubbles, but I don't think you did everything correct. Then I thought about a high content of membran proteins and lipoproteins. Especially multi-spanning membrane protein can fragment and precipitate when heated in Laemmli buffer at 95°C. Or that a high amount of lipoprotein precipitate during the running.
I would suggest not to heat the sample (and use a relativly low protein concentration) in the Laemmli sample buffer, just a 10 min incubation at ambient temperature.
But that is a difficult problem, I have never seen such a effect in a gel.
Best,
Carsten