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I am interested in this kit (ab100663) but want to know for the cell collection what buffers are compatible and what are not. Also, what is the difference between this kit and ab100662?
Asked on Apr 20 2012
Thank you for contacting Abcam regarding our VEGF Human ELISA Kits.
The main difference between ab100663 and ab100662 is the sample types that can be analyzed. Ab100663 can be used to analyze cells and tissues, while ab100662 should be used to analyze serum, plasma, cell culture supernatants, and urine.
Please see the guidelines below for your sample preparation of cell or tissue lysates:
The cell or tissue lysates for use with this kit can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. General low-salt lysis buffers can be used with the following caveats:
1) Avoid using SDS or other strongly denaturing detergents. In general, non-ionic detergents, such as Triton X-100 or NP-40 are best, although zwitterionic detergents, such as CHAPS, or mild ionic detergents, such as sodium deoxycholate will work.
2) Use no more than 2% v/v total detergent
3) Avoid the use of sodium azide
4) Avoid reducing agents, such as dithiothreitol or mercaptoethanols
In general, we strongly recommend that you add some type of protein inhibitor cocktail to the lysis buffer prior to homogenization. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protein inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used, but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated (activated) forms of the protein.
Choices of the method for lysis and homogenization include glass-bead smash, douncing, freeze-thaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no one best method for all sample types, but some are better than others for some sample types. Your choice of method should be made following a brief search of the literature to see how samples similar to yours have been prepared in previous investigations.
After homogenization, spin down the lysates to remove cell/tissue debris (5 min @ 10000 x g or 10 min @ 5000 x g) and save the supernatant. Lysates should be frozen as soon as possible, and stored at -20C (or -80C, if possible). Spin them again before incubating with the antibody array. Determine the protein concentration of your lysates (using a Bradford-Lowry-type assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay.
Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 uL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target for total protein concentration of the homogenate should be at least 1000 ug/mL, but 2000 ug/mL would be better.
Answered on Apr 20 2012