I am doing secretion assay for Proopiomelanocortin (POMC) from N2a cells by using DMEM containing %0.1 BSA, w/o FBS, and by collecting medium in every 30 min. Then I stimulate the cells with KCl. These samples are concentrated by TCA-acetone precipitation. However, I could not observe any band and If I do not TCA-acetone precipitation, samples were getting stuck the stacking gel and separating gel interface.
So my question is that what would be the problem, and does anyone know the secretion protocol of POMC from N2a cells?
1 - after precipitation, in which buffer you eluted your pellet?
2 -Before loading in the gel, have you quantified total protein content?
3 - Do you have a positive control, or a purified POMC in the gel?
4 - is POMC secreted as a free protein or are in vesicles? Then, another centrifugation protocol is needed.
What is your exact protocol of TCA precipitation? Before SDS-PAGE loading I wash my TCA precipitated samples with deio water ( and I do not use acetone).
Thank you Bustamante-Filho, here is answers for your questions;
After precipitation, I tried to elute with 1X and 2X sample buffer. But pellet didn`t dissolve, and I tried to use dH2O. This could be more helpful to dissolve pellet but total result was the same.
I have not quantified total protein content.
I am not have positive control yet but I will have in a month.
POMC is secreted in the vesicles by constitutively, but with the stimulation we expect the increase in the regulated secretion. I am adding a figure that shows this secretion to be more understood.
Thank you Brichta, here is answers for your question;
I used 1:8:1 ratio for sample, 100% ice-cold acetone and 100% TCA, respectively. Precipitate at -20 °C for 1 hr (sometimes overnight). Centrifuged at 15000 rpm (highest speed of our machine) for 15 min. Then washed with acetone and centrifuged again.
Then I also used equal volume of sample and 20% TCA, precipitating on ice for 30min, then centrifuged at 15000 rpm for 15 min, sometimes 10 min. Washing with acetone and centrifuge again.
In both procedure, some samples formed pellet but most of them did not.
Does the washing with acetone affect the result?
I have also one more question. Does FBS should be used in medium for Secretion assay? I read that FBS should be eliminated from sample for good western blot result.
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