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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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RE: protein concentration
Posted by: Saira (IP Hidden, New member, 2)
Date: April 7, 2005 11:14AM
Hi,
I've encountered a problem where the protein samples that I collect with my experiments sometimes have very low concentrations. I use a lysis buffer to extract the protein (about 50 to 100 ul). On other occassions, the protein samples do have high concentrations, but I'm trying to figure out how to make this consistent. I have to use a 0.3 mg/ml collagenase to digest the collagen gel on which the cells are cultured over a short time, and then I proceed to collect the samples for protein extraction. I was wondering if this could be effecting the protein levels. As well, is sonicating a sample for a longer period of time (say 30 s, done 3-4 times) detrimental to the protein content. Any help is appreciated, thanks! saira
Re: RE: protein concentration
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 12, 2005 08:10AM
It sounds you are doing the right thing. However, I would suggest the following: you need to be conssitent in the treatment of all your samples for example if you lyse your cells by sonication, you need to centrifuge and then measure protein concentration in the clarefied supernatant using Bradford, Lowerey, or BCA assays. If you use mechanical lysis like freeze and thaw or glass beads or chemical lysis by a mixture of detergnets in your lysis buffer it has to be done the same for all your samples, at the end of the day if you need to measure protein concentration of samples using either method and compare the protein yield using these cell disrubtion approaches and then you can select the best one according to its protein and non-denaturing effect.
I hope this information is helpfull for you, good luck!
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