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IMG-319 [Monoclonal Antibody to TLR2 (Toll-like receptor 2)/CD282]
I want to know how many mg/ml of this Ab, because I can't find any information of it. But I need to know it, otherwise I can't do the experiment.
Answer :
Thanks for your question about IMG-319 TLR2, As noted on the data sheeet http://imgenex.com/antibody_details.php?catalog=IMG-319, IMG-319 is sold as ascites solutions. Therefore there is not a concentration, but rather a volume and a suggested usage. In an ascites solution, a mg/ml concentration of specific antibody can not be determined. This can only be determined for purified antibodies.
I ordered four monoclonal antibodies from your company a few weeks ago:
TLR1 cat no: IMG-5012, TLR2 cat no: IMG-319, TLR3 Cat No: IMG-315A TLR9 Cat No: IMG-305A I tried western blot on ocular surface epithelial cell lysates using your recommended dilution range using the following protocol. My western blot procedures are as follows: 1. Run 50ug per well of cell lysate on 10% SDS-PAGE 2. Transfer to a nitrocellulose membrane at 100mAmps constant current overnight at 4**C 3. Blocking with 5%(w/v) skim milk in TBST at 37**C for 2h 4. Incubate Primary antibody( IMGENEX) at 4**C for overnight TLR1 Ab: 2ug/ml TLR2 Ab: 1: 500 dilution TLR3 Ab: 2ug/ml TLR9 Ab: 2ug/ml 5. Wash 3 times for 5 min in TBST at RT 6. Add HRP conjugated anti-mouse IgG ( Santa Cruz Biotech, 1:1000 dilution) at 37**C for 1h 7. Wash 3 times for 5 min in TBST at RT 8. Develop with Amersham ECL detecting reagent for 1min, then expose a film from 3min to 10 min in dark room Other than the antibodies, the rest of the reagent all works as I also run western blot on other abs. I am also certain that the above proteins express in my cells based on the positive RT-PCR results and functional analysis (ligand stimulated secretion of cytokines). I would greatly appreciate if you could advice me on how to modify my current protocol. Can you provide me with information on how to optimize my assay, and whether or not RT-PCR data correlates to detectable levels of protein for WB screening? Also, have you any data regarding the use of the antibodies in ocular surface epithelial cells?
Answer :
We have not evaluated these antibodies in Ocular Surface Epithelial cells. I recommend that you use the stated positive control cells (Ramos) listed on the data sheets; and then work out the conditions for their cell lysate. For example, you can run Ramos lysate in parallel with their lysate. Although RT-PCR and functional analysis indicate the presence of the TLRs in you system, it is difficult to know what level these proteins are expressed in your system and whether they can be detected by the antibodies or how much lysate would be needed to detect the TLRs. This is why it is important to use the positive controls indicated, particularly when if you do not initially detect a band in your model system. You may also want to look at the published references using these antibodies.
I looked over the Western Blot protocol. I notice you used a second anti-mouse step. Please note that TLR1 is polyclonal rabbit antibody and you need to use an anti-rabbit secod step for that antibody. If you are obtaining a blank film, then you may want to do different film exposures. If there is background, you might consider titrating the lysate (we use about 20 ug/lane of Ramos lysate).
I want to know how many mg/ml of this Ab, because I can't find any information of it. But I need to know it, otherwise I can't do the experiment. So please answer me ASAP if you can. Thanks a lot.
Answer :
As noted on the data sheeet http://imgenex.com/antibody_details.php?catalog=IMG-319, IMG-319 is sold as ascites solutions. Therefore there is not a concentration, but rather a volume and a suggested usage. In an ascites solution, a mg/ml concentration of specific antibody can not be determined. This can only be determined for purified antibodies.
Keyword(s):
TLR2, TLR-2, TLR 2, IMG319, Toll Like Receptors, Toll-like Receptors
Question : 4
Dear friends, can you provide me some more details about the immunogen for Anti-TLR2, Img-319? Is the extracellular domaine derived from purified non-recombinant human TLR2, or is the immunogen rather a recombinant protein?
Answer :
This antibody was developed against purified partial length recombinant protein. The antigen was obtained from a collaborator about 6 years ago. Due to confidentiality of the project, we can not provide the complete sequence of the immunogen. However, the immunogen contains entire extracellular domain.
Keyword(s):
TLR2, TLR-2, TLR 2, IMG319, Toll-like Receptor, Toll like Receptor
