Anti-CD147 antibody [MEM-M6/1] (ab666)

many thanks for your generous offer! The antibody we require should
have the following properties (as we would like to detect human CD147
from xenografts grown in mice later on):
- human-specific (must not detect mouse CD147)
- must not be from mouse (or directly HRP-conjugated from mouse)
- detect all 4 isoforms (bind C-terminally or on the extracellular
domain closest to the cell membrane, which is also expressed in all
isoforms)
Unfortunately, as far as I can see, ab108308 is the only antibody on
your list which fulfils all these criteria. What would you recommend?
Try the same antibody again from another batch (maybe other users
reported problems or malfunction with this antibody, also?) or go for
another antibody?
I hope we can find a satisfying solution; ab666 yielded very nice and
promising results, however, we need an antibody which fulfils the
criteria stated above urgently to confirm and extend our experiments.
I am looking forward to your response!
Again, thank you very much for your offer and have a pleasant evening!

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. It does look like ab108308 may be the best fit for your experiment, so as requested, I have issued a free of charge replacement of ab108308.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

please find the following protocol, if you need any more specific information, I am glad to provide it!

Dilute protein (12.5 µg/slot) with RIPA buffer Add protein loading buffer (including 2% SDS and 5% beta-mercaptoethanol) Denaturation (and reduction) 5 min at 95 °C --> on ice PAGE at 70-80 V for 3h on 12% gels (I increased the voltage when it took too long) Blotting on PVDF (activation: 5 sec MeOH; 5 min H2O; 15 min blotting buffer) or nitrocellulose (activation: 15 min in 20% MeOH buffer) membrane at 350 mA for 100 min. Ponceau S & wash off in PBS Blocking in PBS/0.1% Tween 20/5% SMP for 1 h Primary antibodies: ab666 & ab108308 1:1000 (1 µg/ml each); shake at 4°C overnight Wash membranes 2x with PBS/0.1% Tween 20 Secondary antibody (HRP-conjugated) 1:3000 (protocol: 1:5000); shake 45 min at RT Wash 2x with PBS/0.1% Tween 20 & 2x with PBS Incubate 1 min with Luminol reagent; development: 10 sec or 60 sec (I sent you an image of the latter)

That is it from my side, again, if I forgot to mention something important, I am happy to provide the information! Hopefully, I do not need to exchange the antibody, as it may be helpful to find splice variant basigin-3, and as a band can be observed in that region, I am very hopeful to get the protocol settled!

Many thanks in advance and have a nice evening!

ANSWER:

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments, and would like to offer a free of charge replacement in compensation (providing the product has been purchased in the last 120 days). In order to arrange this, I would appreciate if you could confirm your order number and date of purchase?

Thank you for your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.

many thanks for your response! I performed a Western Blot to detect CD147 utilizing both antibodies ab666 and ab108308. Although I used the conditions recommended in the data sheet for ab108308 (reducing (and denaturating) conditions; 1:1000 dilution; 12.5 µg protein per lane), I could not detect anything in the range expected (and confirmed by ab666). Please find a copy of the blot attached. To the left of the marker protein ab666 was used, right of the marker ab108308.

As you confirmed, ab666 detects an epitope on the second extracellular loop and therefore only splice variant basigin-2 (and basigin-1, which is retina-specific), and ab108308 detects a C-terminal epitope (and therefore all 4 known splice variants). However, as you can see, I achieved some decent results for ab666 detecting both the highly- (~50-60 kDa) and low-glycosylated (~33 kDa) forms of CD147, but nothing in that range for ab108308. The only very slight bands I was able to detect might (from the protein mass) be attributed to basigin-3 (~28 kDa).

Therefore, I turn to you with the question what I might improve to improve my results and detect the same bands I got with ab666.

ANSWER:

Thank you for contacting us.


I am sorry to hear that of this problem. No or low signal can be the result of a number of factors. As this product is a tissue culture supernatant I would suggest that prehaps increasing the antibody concentration may help. If you could send me the protocol that you have used I may be able to offer furhter tips as well.



This product is covered by our Abpromise guarantee.If we cannot remedy this issue and this is a product that you have purchased within the last six months, we will replace or refund it under our Abpromise guarantee, if being used according to specifications listed on our datasheet.



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

we purchased anti-human CD147 antibody clone MEM-M6/1 (ab666). However, due to different nomenclatures, I am not sure what epitope, and what isoforms / splice variants in particular, this antibody detects. With respect to the attached paper (Liao et al. 2011; Figure 1E), could you please specify which isoforms should be recognized by this antibody? Another paper (Yu et al. 2008), in which the crystal structure of basigin-2 was resolved, might also be of aid and emphasize difficulties regarding the nomenclature of domains.

Many thanks in advance and a pleasant weekend!

ANSWER:

Thank you for contacting us.

The epitope should be located within the N-teminal Ig domain D1, which is the more membrane-distal domain, thus IgC2 domain as called within the paper mentioned.The most information about epitope of thios product is contained in the following paper:Koch C, Staffler G, Huttinger R, Hilgert I, Prager E, Cerny J, Steinlein P, Majdic O, Horejsi V, Stockinger H: T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density. Int Immunol. 1999 May;11(5):777-86.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

We are interested in buying from Abcam the products [MEM-M6/1] (ab666) and [MEM-M6/6] - Low Endotoxin (ab119114).   In our experiments it is very important to know  the isotype of the light chain. May I please ask you if you know the above information.        

ANSWER:

Thank you for contacting Abcam regarding these antibodies.

I have spoken with our laboratory regarding the light chain isotypes of ab666 and ab119114. Unfortunately this information is not available.

Please do not hesitate to contact us if there is anything else that we may do to help you reach your research goals.