I have extracted the protein sample using Tris phenol method, and after extraction I have resuspended the sample in Tris Hcl...The extracted protein sample have a concentration of 10ug/ml through Bradford assay...
Then I loaded, (20 ul sample +30 ul loading buffer ) in one well and (25 ul sample + 25ul loading buffer) in another well..
But I am unable to get the bands after running those samples in SDS PAGE.
I am attaching the gel picture..
Please help how can I get proper bands.
Dear Swarnali Das
It seems that you loaded no more than 0.25 ug of protein in the well. It is too low for Coomassie detection. Even if you had there only one protein (which is not probable), it would be barely visible. Possibly you need to concentrate the protein sample about 100 times (e.g., by centrifugal filters). Optionally, you can use a more sensitive dye, such as SYPRO Ruby Protein Gel Stain (with fluorescent detection).
You need to include controls. Controls are more important that samples. Without a MW ladder or other standard on your PAGE, you can't know whether the extraction failed or your PAGE failed. Always include controls.
Also for any purification, it's best to test at every step. The phenol extraction method has two main steps. So save a sample of your aqueous extract to run on PAGE along with the final precipitated product (and a standard or two). That way you can determine whether maybe your extraction method worked at least part of the way.
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