Hello,
Please can you help me. I need to concentrate cell media supernatant and detect GROWTH FACTORS protein with ELISA method, wich one you recomend?
Hi! one of the easiest way to concentrate something is in a vaccum chamber. Some are equiped with a centrifuge so that the concentrate goes down in the tube at the same time it is concentrated. I hope it is useful this idea!
J
Hi,
I will suggest you used first ultra-filtration method(Amicon) using membrane(10-30kDa) depending on your protein MW cut off.Thereafter you can use a viva spin(PALL Microsep advance centrifugal device) and centrifuge till your desired volume.It becomes more concentrated as you centrifuge.You can run check your supernatant on native-PAGE whether it leaked or not.
I hope this will help you.
All the best!
I have experience using Amicon Ultra Centrifugal filters. which come with different kDa cutoffs and volume sizes. You can try them. They are easy to use and are good.
http://www.merckmillipore.com/IN/en/life-science-research/protein-sample-preparation/protein-concentration/amicon-ultra-centrifugal-filters/YKSb.qB.GXgAAAFBTrllvyxi,nav
I have analyzed urinary proteins (please see file; urinary alb determination), and have found that urinary-biotinidase excretion is occurring early in the IDDM (please see file; Dr. Terentyeva urine-BIN). Urine has been concentrated by cellulose-dialysis tube with dry-powder of PEG 20,000 (please concentration should be stopped before dry up), and we have discovered urinary biotinidase, which is distinctively different from serum biotinidase and milk biotinidase (please see file; HepG2 Fucoidan). Urinary or kidney biotinidase has N-terminal sequence of AVPPQVGNGQEGQTN-, and milk or mammary-gland biotinidase has N-terminal sequence of FPSYVAVTKV VPPYG- as determined by the Edman degradation method. Fetal/inflammatory-biotinidase has N-terminal sequence of AHTGEESVADHHEAE- (Cole H, Reynolds TR, Lockyer JM, Buck GA, Denson T, Spence JE, Hymes J and Wolf B: Human serum biotinidase. cDNA cloning, sequence, and characterization. J Biol Chem 269: 6566-6570, 1994 as determined by PCR-DNA sequencing method).
Please do not use non-quantitative ELISA method, and please use the reliable Edman degradation method (PDMD method in the file "HepG2 Fucoidan).
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