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Inquiry: I want to order this product but need to know if it can be used on tissue homogenate of rat.Pls let me know as soon as possible to order the product
Asked on May 07 2012
Thank you for contacting us.
I have contacted the lab and received the following information:
While ab100769was neither designed nor validated with any lysate samples, the kit is very accommodating to many different samples types so lysates will likely work. If the customer decides to test this kit with lysate samples, we recommend diluting the samples at least 5-fold with Assay Diluent B to minimize any effects of the detergents in the lysis buffer. The samples may need to be diluted further but this would need to be determined by the customer empirically.
For the original lysate, the customer in general would want to aim for at least 1 mg/ml of total protein, preferably more, to achieve a loading concentration of 50-500 ug/ml after dilution. Again though, this range should just be used as a starting point and the optimal concentration would need to be determined by the customer empirically.
For preparation of the lysis buffer, please see below forsome general guidelinesalong with some basic tips for sample preparation.
In brief, a lysis buffer must meet the following specifications:
A) has relatively low salt content (700 mM or less)
B) does not contain sodium azide
C) does not contain >0.1% SDS
D) does not contain >10 mM reducing agents (beta-mercaptoethanol or dithiothreitol)
This would include any buffers used for immunoprecipitations, including RIPA buffer.
How do I make cell or tissue lysates for use with an ELISA kit?
The cell or tissue lysates for use with anAbcamELISA 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 protease inhibitor “cocktail” to the lysis buffer prior to homogenization (e.g. ab65621 (https://www.abcam.com/Protease-Inhibitor-Cocktail-ab65621.html). Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary,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 ELISA kit. 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.
Since tissue and cell lysates have not yet been specifically tested with this kit, you can use our testing discount program in that case: For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:
The testing discount program is for customers who like to use anantibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.
This programapplies to ELISA kits and other kits we offer.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on May 07 2012