Rat VEGF-A ELISA Kit (ab100787)

The cell or tissue lysates for use can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. Other general low-salt lysis buffers can be used with the following caveats:



1) Avoid using SDS or other strongly denaturing detergents (That is why we would not recommend RIPA buffer).

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 protease 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 protease

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 -20°C (or -80°C, if possible). Spin them again before incubating with

the antibody array. Determine the protein concentration of your lysates (for example the

bicinchoninic acid (BCA) 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.

The BCA total protein assay will be fine with our lysis buffer. In fact, we use it all the time in our lab to determine the total protein in lysate samples. To my knowledge, the Bradford assay is the only total protein assay which is http://en.wikipedia.org/wiki/Bradford_protein_assay. You may want to contact the supplier of that kit if you are having trouble with the BCA assay.

Thank you for contacting us.

The concentration of the HRP-streptavidin reagent is considered proprietary information by the developer of the kit. I am sorry we could not be of more help.

Please do not hesitate to contact us if you have any other questions.

I am happy to hear that the new kits are working well for you! Please let me know if there is anything else I can do to help.