I want to measure serum Ghrelin levels in mice samples. I collected serum about 3 weeks ago, and froze it at -80 degree. I only received the ELISA kit 2 days ago. The kit says the sample should be acidified to 0.05N with HCl after collection, and kept at -20C for later use.
May someone with some experience in Ghrelin analysis advise me if its now a 'non starter' to go ahead with the analysis having made the mistakes of not acidifying and keeping at -80C. Do you think it has gone all wrong already? I really don't want to waste the kit because I had trouble getting it, and its a 'one time use'.
Dear Stephen:
I haven`t too much experience about ELISA analysis..... but I can give the mail of a friend of mine who has more experience than me... Paul Martin Coen
is it an ELISA for total ghrelin concentrations or specific for active acylated ghrelin?
Hello Stephen,
I am doing ghrelin ELISA.
The general method is, when you collect blood immediately add pefabloc (available with sigma), its a protease inhibitor, then centrifuge and collect serum. Make a 0.5 N HCl stock and add with serum such that the final concentration is 0.05N.
This is because acetylated ghrelin (active form) is easily degradable therefore the serum is acidified. I store the processed serum at -20 C.
If you have to estimate acytelated ghrelin then you have to follow this method strictly, for unacytelated ghrelin acidification is not required.
I suggest you to collect the blood once again if you did not sacrificed the animal.
If you need any further clarification please feel free and let me know..
TIRU
Thank you for the coming responses! Its the Active form I want to measure. (the kit, though, is for both active and des- octanoyl forms)
TIRU, so what does extreme cold temp (-80 C) have on the active protein integrity?
For leptin ELISA, i store serum at -80C. Protein integrity will be there, but in your case its acetylated protein, so flind can acetylated protein can be stored at -80C. Or just use a single well to test the condition of your acty ghrelin sample.
its a hunger hormone.. my point is what other parameters we may can check related to Gherline can i get some more related parameters?.
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