Hi, I need to check the amount of a certain protein secreted in conditioned media. What I have done so far is plated equal number of cells and then collected equal volume of media. Following this I have done a coomassie staining or Ponceau staining of the blot.
Please let me know if you have any suggestions. Thanks a lot.
Hi there,
I am also looking for the amount of a secreted protein in conditioned media. Instead of staining the gel/membrane with coomassie or ponceau in the western blot I use protein specific primary antibody in blocking solution overnight then appropriate secondary HRP-antibody for an hour, then a ECL kit as substrate before developing. I use GAPDH as the housekeeping gene and incubate it afterwards on the same membrane.
I have also used a pre-coated ELISA kit which allows you to do a more accurate quantification on your conditioned media, they are really easy to use as well.
Hope this is helpful!
Hi Neha,
you can normalized the supernatant with any protein estimation method, BCA, bradford if you are doing a blot. For housekeeping we used GAPDH, Actin as non-secretory house keeping, if you are looking for secretory protein control - its cell type specific.
hope this helps & I got your question right.
- Anand
Hello Neha
I advice you run the supernatants and cell lizates from the same cellular population for western blot. Then, normalize the secreated protein with housekeeping protein (b-actin or GAPDH) from cell lysates. Therefore,The secreted protein is detected from approximately the same number of cells and the same volumen of media.
Best regards
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