We are extracting protein out of the milk fat globules. After extraction we tried to quantify total protein concentration in our samples but unfortunately our final protein yield is extremely low and it is under the limit of quantification of the traditional bradford test. We know that we are indeed extracting protein because we are able to see specific proteins in Western blot but unfortunately we are not able to accurately determine total protein concentration because for most of the samples it is under 500 ng/microL. Do you know any specific technique or kit that could help us to detect protein in such a low concentrations?
Thank you in advance for your comments.
Maybe the lipid debris is interfering with your quantification. You can try the CBQCA Protein Quantitation Kit, which is a very sensitive assay capable of detection as low as 10 ng of protein per mL. This is well suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.You can find it Life technologies and it is really easy to use.
http://www.lifetechnologies.com/ca/en/home/life-science/protein-expression-and-analysis/protein-quantitation/solution-based-assays-protein-quantification.html
I hope you'd find this useful.
Maybe the lipid debris is interfering with your quantification. You can try the CBQCA Protein Quantitation Kit, which is a very sensitive assay capable of detection as low as 10 ng of protein per mL. This is well suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.You can find it Life technologies and it is really easy to use.
http://www.lifetechnologies.com/ca/en/home/life-science/protein-expression-and-analysis/protein-quantitation/solution-based-assays-protein-quantification.html
I hope you'd find this useful.
Hey there!
Thanks a lot, that is exactly what I was looking for, so thank you very much! Have you used the kit before?
Hope that you are doing well in Canada!
Hi you may use the BCA assay.
http://www.piercenet.com/product/bca-protein-assay
Be careful of nanodrop though, because some buffer components can give a false high signal.
Hello,
based on your description of very low protein concentration, I would suggest ELISA detection. It's a bit time consuming method but after some optimization steps we were actually detecting less than 1ng/mL concentration.
The answer is very simple. You have a solution of 10-500ng/ micro liter. Now in order to take it within the limits of the assay, simply concentrate by putting it in a dialysis bag and suspend it in solid sucrose in a beaker. Let out all the water. Estimate the protein and convert it to the concentration of the original solution if required.
Hi there,
in a sense i would agree with Jai. Why don't you also perform a concentration reaction with acetone for example, and then do a normal Bradford assay. When you get your concentrated quntified protein, go back using your dilution factor and calculate how much protein you initially had. That's of course if you are on a low budget because as some people have mentioned here there are kits that allow you to quantify proteins in small amounts.
This aproach by BIORAD may also bof interest to you:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5576C.pdf
Best wishs,
Nikos
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