hello,
i extracted protein from PBMC's and after applying BCA assay on 2 samples the protein concentration was 400 ug/ml which means 0.4 ug/ul so does western blot work with these quantities?
any suggestion?
thank you.
Hello Ayah
In Western Blot for a complex mixture of proteins you need to load about 15ug to 30ug of protein. If you are loading purified protein then 0.1ug to 2ug protein concentration should suffice.
Thank you.
Dear Ayah!
Please You look at this publication:
https://advansta.com/wikis/preparing-samples-for-western-blot-analysis-protein-quantification/
Bicinchoninic Acid Assay (BCA)
BCA is similar to Lowry, except bicinchoninic acid (BCA) is used instead of the Folin-Ciocalteu reagent. After reduction of Cu2+ ions, two molecules of BCA chelate with each Cu+ ion resulting in formation of an intense purple color that absorbs at 560 nm.
BCA is as sensitive as the Lowry method and works well with protein concentrations from 0.5 μg/mL to 1.5 mg/mL. Although detergents do not interfere as strongly as in the Lowry method, other contaminants can interfere with the reaction. In addition, aromatic amino acids can influence the reaction. However, at higher temperatures (37-60°C), peptide bonds can also contribute to formation of the product. Therefore it is recommended to perform the reaction at higher temperatures to increase the sensitivity and decrease the variability due to amino acid composition.
If the protein is pure or almost pure, you should be able to detect down to 100 pg of protein using old school hrp. However, you can detect less protein using chemiluminescence-based detection methods.
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