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problem in Protein qunatification : mice carotid artery

problem in Protein qunatification : mice carotid artery - Biochemistry Forum

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Old 08-29-2011, 07:39 PM
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Default problem in Protein qunatification : mice carotid artery



Hai,

I am doing the rsearch in cardiovascular system. In my project, I am trying to qunatify the protein content from mice carotid artery. My problem is, After quanitfication, when I ran the gel, I didnt get the equal band size for all the sample ( with commasiev staining as well as in the Western blot)...

I have used the 2x SDS buffer for lysis the tissue and then centrifuged @ 14000 rpm for 15 min. After that I took the supernatant for the protein qunatification. I did the protein quantification with BCA protein assay then based on the value I have loaded 10 microgram (Protein) of sample to each well.

I have tried various steps and repeated 3-4 times ... I feel there is a problem in lysis buffer...!!!! I want to know the buffer which I am using (2 x SDS) is correct or not.....!!! I need some idea for you to solve this problem.

with Regards,

Saran
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Old 09-02-2011, 12:18 PM
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Default Re: problem in Protein qunatification : mice carotid artery

If you have a scanned picture of your Blot, post here so that we have better idea.

I like to know
1. do you boil the sample before protein quantification,
2. did the sample still viscous while you taking it for measuring.(if viscous add little more lysis buffer or boil for more time ). Make sure it is not viscous while you measuring protein concetration.
3. Some time different samples from different animals or tissue show variation in protein
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