Hi community:
I was wondering if you could give me more tips on the following.
I am looking at cytokines levels of lungs exposed to an allergen. Specifically, I am looking at intracellular cytokines, from lung homogenates. So I homogenated my lung with a buffer containing protease inhibitor, and run my cytokine assay which is a bead multiplex assay using flow cytometry.
Now i want to normalize the results given in pg/ml to mg per protein found on each sample.
I selected the BCA assay because is compatible with my buffer, and I am also using the same amount of volume (25 ul) from my sample than the volume I used to perform the cytokine assay.
My questions are the following:
-What dilution should I use for lung homogenates to run my Pierce BCA protein assay? Should I dilute my sample even more than the dilution I used to homogenize it and run the cytokine assay?
-My lungs contained a good amount of blood, because the homogenates are pink, but will the hemoglobin interfere that much in my BCA assay??
Ivon Moya Uribe ,
There is no exact answer. You will need to test a few dilutions (I usually go 1:10, 1:100, 1:1000, 1:10000) just to see where samples would fit within the standard curve. Then once you find one that will be within the middle of the curve (ideal) you dilute the rest of the samples down and do a full BCA assay.
Hi Ivon,
Yes, I agree with Shawn Krueger, there are no exact dilutions you should use to calculate the conc. of the unknown samples. It depends on the fitting of your result in the standard curve. Based on a preliminary experiment you should adjust dilution such a way that the final result should fit within the standard curve.
Also, In BCA assay, the color developed due to the reduction of copper ions, so the assay is highly sensitive to the red-ox state. In your case, the iron in the hemoglobin may produce some interference with your calculated result. Though, you can clean up your sample by immunoprecipitation or by using a anti-hemoglobin antibody.
You can follow this link to clean your contaminant cell lysate for BCA analysis. http://www.biotech.cornell.edu/sites/default/files/uploads/Documents/Proteomics_protocols/Protocol%2002_Protein%20precipitation.pdf
Good Luck
Prasenjit
Prasenjit Mondal thank you so much for the info.
Also could you give me your advise on this: if I need to dilute my samples for the BCA assay that are already homogenized in their buffer , do I keep diluting them using more of that same buffer?? (assuming that my buffer does not contain any interfering substance for the BCA assay), or do I use a different diluent like RO water or something of that sort?
Thank you!
Hi Ivon,
Yes, you can dilute your sample in the same buffer if it doesn't contain any interfering substance.
Good luck
This strongly depends on the ell numer harvested in you experiments. However a titration series as mentioned above should work (e.g. 1:10, 1:100, 1:1000, 1:10000).
Update on results :
I performed the BCA analysis using different dilutions. All my OD measurements from my samples at different dilutions where within the standard curve range, except for the highest dilution (1:400) that was below the cut off. I sent this results to tech support from the company where I purchased the BCA kit and they thought there was no hemoglobin interference or other components that could be altering my results.
Despite this, i still obtained results on my samples that didn’t follow the same pattern than the cytokine measurements results.( My negative controls that had almost no detectable levels of cytokines , had the same amount or higher levels of total protein amount compared to the treatment grouped that showed higher levels of cytokines.)
If anyone would like to give their thoughts on this please feel welcome to comment.
I would appreciate any possible feedback you think it would be worth to mention.
Thank you for all your previous answers
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