After qPCR analysis of HUVEC lysate from my experiment, I found upregulation of several chemokine mRNA that I would like to validate via western blot. Due to the nature of my experiment, it would have been impractical to collect the media and analyze secreted chemokines via ELISA as is generally recommended. I did not apply brefeldin A to inhibit secretion as I had not initially intended to examine chemokines. My experiment would be expensive and time consuming to replicate, is it possible for me to quantify my chemokines of interest using this total lysate?
If I were you, I would just repeat the experiment. Let me explain why.
1. If you are talking about the cell lysate you used for qPCR, then probably not. They way you lyse cells for RNA analyses and protein analyses are generally different. You will have to lyse the cells in Laemmli buffer/RIPA buffer etc. to do western blot. The lysis protocols/reagents are usually optimized to minimize contamination from other cell components. Hence, using one lysate for estimating RNA/protein is not possible unless you used a specialized reagent that allows for estimation of both. Even then, it wouldn’t be ideal. You may be able to detect intracellular cytokines in the absence of secretion inhibitors but the estimates are not going to be accurate.
2. Chemokines are secreted once synthesized. Not all chemokines are expressed at the same time. So if you do an ELISA on the sups at 24/48 hours post treatment, you may be able to pick up differences in all relevant chemokines due to accumulation over time. When doing western blot, you will have to do a time course (lyse cells at different time points) to get the same results.
3. I understand it is going to cost money and time to repeat the experiment. But if decide to do WB, you would end up having to buy both primary and secondary antibodies to detect all the chemokines you are interested in. Provided you have 5 chemokines that you are interested in and that you can use the same secondary antibody for all of them, this would still set you back $1500-1800 (approximately $300 per antibody). A multiplex chemokine kit that you could use on cell culture sups/lysates might come out cheaper.
4. Since your results are not going to be robust (as your experimental setup was not optimized), you would end up having to repeat it and chances are high that this time, your results might be different.
I would follow Julie Joseph's suggestion.
It's unclear where these lysates are coming from, and if it's the one that you isolated your RNA, the protocol would have eliminated the protein.
The only thing you can do is if you stored original lysates correctly (at least -80C). And the only thing you will be able to do is to determine if the protein is present. You won't be able to quantify (you sort of can if you use a housekeeping protein as a normalization). And there are processing of the lysate to consider.
Julie Joseph Shen-An Hwang
thank you for the answer. I should have specified that I lysed a different group of cells from the same experiment with RIPA and stored them at -80 to perform westerns later on. I have used this lysate to analyze several other genes and have gotten good results, so I know the protein is good quality.
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