Hi researchers,
My colleague passed me same serum samples (300ul each tube). He thawed it from stock and his operation was normative. I wanted to aliquot 50ul from each tube but I didn't know I need to put the serum on ice until half an hour later.
I want to do ELISA with the serum, will my aim protein degrade during this half an hour?
After my colleague told me I should put the serum on ice, I do the aliquot on ice for the rest samples. Hope this won't affect too much to my result.
It is highly depended on the protein, but half an our should generally not be a problem.
Hi there,
It shouldn't be a problem but as mentioned above it is actually protein dependent so if you want to push further into this direction just run two experiments in parallel : one with the sample kept on ice all the time and the other with your sample left at room temperature for a while. If the results are not significantly different, your protein is stable if they are your protein might be affected by resting at room temereature..
It is highly depend on the protein sample. For my experience, human protein is less stable and can be degraded in room temperature, but bacterial protein can last long even in Room temperature,
Hi Shuaichen Liu,
as our colleagues said before it depends on the sample (eg. what additives are present eg. EDTA, heparin, citrate etc.). It also is highly dependent on what protein you wish to test for. Some hormones for example will last some hours only whereas antibodies are quite stable, even at room temperature.
If you are interested you can read more about this topic here:
http://cebp.aacrjournals.org/content/15/9/1578.full
http://www.sciencedirect.com/science/article/pii/S0009912001001965
Best wishes,
Christiane
Keep in mind how you assay for your protein. If it is an enzyme or an injected ligand for example and you use SDSPAGE to assay then it may look fine on a gel but it could have undergone conformational changes that cause inactivation which would need to be assayed by activity methods to determine stability.
Hi Shuaichen Liu,
do you know the protein composition of your serum? Are proteases present in this serum?
First of all, it depends on the ambient temperature. If it's 15-16 degrees centigrade, you have less problems; if it is 30 degrees, then everything changes. However, 30 minutes is not so much, but always remains the suspicion that something might not have worked when the results are a bit chaotic or abnormal. In this last case, most likely you will need to repeat everything to placate your consciousness as researcher.
It is highly depended on the protein, but half an hour should generally not be a problem, but if you have proteases in the ambient, is better work on ice and using gloves
For most proteins and assays, 30 min at RT should not affect your results, but there are more sensitive proteins and/or samples.
It is better to work on ice with biological samples, if unsure of the denaturing property of a specific protein. You may check literatures to be sure of. Good luck.
As serum is biological sample and it contain heat labile proteins...so its necessary to maintain low temperature while working with such samples....its sure that ur results will definitely improves when u maintain the low temperature while working with protein samples
Don't forget that serum spends all of its time at 37 °C, until you collect it. The proteins are not heat labile at room temperature. For ELISA even some protein degradation usually has no effect, so long as the epitope itself is not cleaved. Also, it is possible your serum was collected with various added inhibitors that would reduce the effect of certain proteases, such as metalloproteases. It does depend on the target, but I'm going to say you are alright. Next time, put the sample on ice, though.
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