serum separation: transparent gel prevents serum collection! - (Nov/17/2010 )

Hello! For the first time I am sampling animal serum to test with ELISA for the presence of a protein. The protein I want to test is rare and unstable and I was afraid to lose it. Therefore I didn´t wait long for the blood clot to form, but I centrifuged (2.500 rpm, 5 minutes) after some minutes instead. I could mostly separate serum, but sometimes (20% of the samples more or less) after centrifugation the serum was separated but it was in the form of a transparent gel instead of liquid (therefore impossible to pipette). Why does this happen? is it because I didn´t wait long enough for the clot to form?
And what happens if I centrifuge before the clot forms, do I get also the coagulation factors together with the serum? (I am using plain glass tubes, with no anticoagulant and I am collecting 3 ml of blood).
Thanks for any help ypu can give me, as you see I am a very beginner in this field!

I would suggest testing both serum and plasma. Centrifuge edta or heparin tubes immediately after collection, aliquot and freeze. For plasma allow the clot to form completely (you may wish to use the gel separator tubes) centrifuge and aliquot and freeze. Use the aliquots once then discard. You may also wish to test the 'stability' by running a thawed sample over time.

I am making an assumption that the protein is stable frozen and does not change upon thawing. If this is the case then you will have to explore diluting the sample in some type of buffer to preserve it. There are several additives and buffers on the market.

If you are running an elisa then what are you using for your standard or calibrators and what matrix is it in? Your samples should have about the same stability (with azide/thimerosal/etc).

Sorry, my first line was for serum not plasma. Plasma has the clotting factors and is the supernatant once heparin or edta tubes are spun.