Hello everyone,
I am pretty new to ELISA and I have been thinking of conducting a ROMO1 quantitative sandwich ELISA with NHDFa. I am planning on having three samples in triplicates however in our institute we are using a small device to culture our cells on and therefore I can only have a maximum amount of 50.000 cells per sample. Obviously I need to run an optimization series with my unknown samples to find a dilution value which lies between my min/max OD of standards. However I am not sure if 50.000 cells/ml per samples would be enough to get signals. And since these kits are expensive I cannot afford to get more than one.
What is the "theoretical" min. concentration of cells that can be used in such an ELISA? I could not find anything related to my question in literature...
I would very much appreciate your help
Thanks :)
Hello Ipek, I do not think the minimum concentration of cells is as important as the actual concentration of the protein extract which should be 1 mg/mL. 2 mg/mL is not too much too. So do not bother your head too much. Have a go at these tips.
General tips for ELISA sample preparation:
- Serum, plasma, cell and tissue extracts are typically diluted by 50% with binding buffer.
- Total protein concentration of homogenate should be at least 1 mg/mL. However, 2 mg/mL or more would be better.
- The collected samples can be kept for different periods: 48 hours (2-8°C), 1 month (-20°C) or 6 months (-70°C).
- The protein concentration of your lysates can be determined by a total protein assay not inhibited by detergents such as the Bicinchoninic acid (BCA) assay. The volume of each sample can also be normalized to deliver the same amount of total protein for each assay.
- I have attached a copy of ELISA Handbook for you. All the best.
Hello, thank you so much for the answer!! Helped me a lot.
Do you also happen to have/know a good freeze/thaw protocol for cell lysis?
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