Hi everyone,
I am doing western blot for POMC/ACTH secreted from AtT20 cells. The problem is as seen in the picture I added. First 3 lanes are for lysates and not a problem. But the next lanes are for secreted mediums.
This huge blue area appears after staining the gel with coomassie dye, and also in membrane after staining with ponceau S.
To concentrate the protein sample, I precipitated them with acetone, then resuspended in sample buffer. Although I tried to precipitate 100 ul, 200 ul, 300 ul samples and dissolved in 30ul , 50 ul of Sample buffer, the result is same. The amount of protein did not change the result. I also used 4-12% and 4-16% gel, changed the recipes of gel according to many protocol. So, whatever I do , they are not separated. How can I overcome this problem?
Hi
I suggest that you should load a lower amount e.g 10 -15 ul .
I hope it will help
Hello Merve,
I think that your problem is the high amount of protein in secreted samples. Altough you precipitate with acetone 100-300 uL of protein, for SDS-PAGE the protein concentration in the sample must not be exceed 50 ug, if not several problems of resolution could appear. After acetone precipitation and washes with 80% acetone , you could resuspend the pellet proteín in SB 1 X, and then quantify the protein for load 50 ug of each sample. The voltage in electrophoresis and tampon also could be a important factor for a good resolution in SDS-PAGE.
Good luck!
Dear Merve,
As secreted samples contain more amount of protein, it is better to reduce the volume of sample to be loaded (max- 10-15 ul (5-10 ug protein)
I read many paper about concentration but none of them says about its amount. I added one of them which is most relevant what I`ve done. It says that pellet dissolved in 72 ul 1X sample buffer. That`s it. It does not say how much was loaded.
Once I measured the concentration after precipitation, and I had to dilute it 1:300, so If I want to load 5-10 ug protein, it has to be diluted too much, since the concentration is too high. And I am not sure such amount will be enough to see my interested protein.
Dear
Based on your gel picture, it seems, concentration of protein if too high (around 500ug to 2 mg). it is better you dilute the sample, run the SDS PAGE.
If you varied detection......please follow silver staining protocol, because, it can detect up to ng level.
I hope this information is sufficient for your quiry
If you need silver staining protocol- please send your e mail ID
thank you
Concentration is too high, maybe u can take this opportunity to run a gradient dilutions on a 15 well gel, for example, 1:100 dilutions followed by 1:300 dilutions, then 1:600 dilutions, and so on. This helps with your future loading guideline.
The big 72kDa blob is serum proteins. If your question is about secreted protein from cells, just wash the cells in serum free medium and incubate the cells in serum free medium. As long as you use FBS containing medium you'll see the big blob around 72 kDa. Good luck..!!
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