Rat TNF alpha ELISA Kit (ab100785)

Yes, that lysis buffer will be good, the 150 mM sodium chloride, 1.0% Triton X-100, 50 mM Tris pH 8.0.


Here are some general guidelines.


Avoid using >0.1% 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. 


Use no more than 2% v/v total detergent  


Avoid the use of sodium azide 


Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols 


We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to homogenization.

Thank you for contacting us. The cell lysis buffer does not contain any protease inhibitor. Please let me know if you have any further questions.

I can suggest the following general tissue lysate preparation procedure to prepare samples for both the kits.




Tissue lyste samples can be prepared using most conventional methods, for example with RIPA or other formulations.

Please note the following guidelines on lysis buffer composition:
1) Avoid using >0.1% 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 using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols

We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to
homogenization. Most general biochemical supply companies including stock a wide variety of these products. 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 forms of the protein.
Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw,
sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these.
There is no best method for all 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, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10
min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as
possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any
immunoassay. Next, determine the protein concentration of your lysates using a total protein assay
not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of
each sample used to deliver the same amount of total protein for each assay.

Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.
Since different cells and tissues may contain different amounts of protein, as starting point, we
suggest using 500 μL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this
based upon your results. Your target total protein concentration of the homogenate should be at least
1,000 μg/mL, but 2,000 μg/mL or more would be better.

Thank you for your enquiry.

Please rest assured that there are no hazardous components in this kit according to Japanese law, and is safe for use by customers in Japan.

Have a nice day!

Thank you for your enquiry.

The lysis buffer (item J) is used to prepare the cell and tissue sample. There is no mention on how to prepare your sample in the Protocol Booklet as most laboratories would have their own protocols and methods for doing so. We do recommend starting with 1mg/ml, preferable 2mg/ml volume (sample in lysis buffer) of sample. Please bear in mind that this is a starting point and the optimal concentration needs to be determined empirically.

You may also use your own lysis buffer. As a guideline, 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)

I hope this information will be helpful.