Hello everyone. I am doing immunization. after doing ELISA, i left serum collected from mice at room temperature for more than 10 hrs. Is there any chance that it is contaminated or my Antibody (anti-Agr2) is degraded?
Serum does not contain free proteases but the only way to know the answer is to use your sera again. If they are not sterile, I think 10hrs at RT (23°C probably) is not a long time for bacterial or yeast growth. Good luck
Hi Sehar, I agree with Paola. Sera has more proteinase inhibitors than proteinases.
If you have microbial contamination of your sera, it should be obvious by being cloudy.
I would like to express my opinion. Serum is stable body fluid against protease due to alpha1-antitrypsin and alpha2-macroglobulin, and stable against microbe invation due to immune system (please see JMBT Alopecia). Milk is also stable against protein degradation due to scanty of protease, and stable against bacteria due to lysozyme. But, tissue is not stable. Tissue invation by microbes has been found to be very quick; i.e., ra liver is quickly invaded within 3 hours at 18 degrees of Centigrade after the death as assessed by determining biotin decrease (unpublished observation).
On the other hand, PDMD (protein-direct-microsequencing-deciphering) method requires fresh serum; i.e., protein binding rates to glass fiber using EDC decrease immediately possibly due to thiol-thiol reaction occurred in serum. This indicates that serum is labile chemically.
Therefore, biochemical handling of serum and tissue should be promptly performed.
By the way, quick handling of serum and tissue homogenate is required for chemical PDMD method to perform. PDMD method requires diluted samples; i.e., protein concentration should be adjusted to 1.0 mg/mL. Then, HPLC-SEC protein determination method is indispensable, which is rapid to determine the protein concentration within 3.5 min, and reliable (see files; serum prot deter, HPLC-SEC protein determination method, and prot deter Summary). During the HPLC- protein assay development, we have discovered the adequate serum-dilution method; i.e., neutral 0.1 M phosphate buffer (pH 6.8 or pH 7.0) containing 1 mM EDTA and 10% (v/v) glycerol, which is stable solution against microbial contamination possibly due to EDTA.
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Dear
Your sample could be degrade after long storage due to microbial contamination or instability of some amino-acids. Proline-containing peptides are known to be among the least stable peptides since they undergo rapid degradation in serum samples.
To obtain a stable serum, uncentrifuged clotted blood should be stored at 4 °C and processed within 6 h.
Please finf more information in the following publication: Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma. Nancy B.Y. Tsui, Enders K.O. Ng, Y.M. Dennis Lo. Clinical Chemistry Oct 2002, 48 (10) 1647-1653.
Hoping to be helpful
Marius
Thankyou so much for your help. much appreciated. Dear Stefannson, Serum looks pretty clear and i used it gain. results were ok. Thanks again everyone!
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