Anti-HSV1 + HSV2 gD antibody [2C10] (ab6507)

Thanks for your reply. Here are the details of the protocol that I followed:

Guinea pig fibroblast cells were infected with a recombinant virus with the gene for HSV2gD (from strain 333), HSV1 strain 17Syn+, and HSV2 (strain HG52). The positive controls of HSV were harvested 24 hours post infection; the recombinant virus were incubated until the monolayer was at least 80% infected. The monolayers were harvested using dissociation mix and, after heating at 95 C for 5 minutes, 30 ul of cell lysates were loaded onto a polyacrilimide gel. The gel was transfered to a nitrocellulose membrane (using markers to confirm that the transfer worked). The samples transferred to the membrane included mock infected cells, the backdrop virus, two recombinant virus samples, HSV1 and HSV2. I followed protocol provided in the Western Breeze Kit (anti-mouse) and we have had success with this kit in the past. Again, the dilution used for the HSVgD primary Ab was 1:500. The membrane was incubated for 1 hour with primary Ab the first experimental run. When I repeated the assay, primary Ab incubation was for 3 hours. Both times I performed this Western Blot assay, bands were visible after 1 hour of incubation with the chromogenic substrate only in the lanes containing the recombinant virus samples. In both lanes, a band at 55 KDa and a slightly less intense band just below it were visible on the membrane. No bands were visible in the negative controls, nor in the HSV1 or HSV2 samples.

We have a few concerns with these results related to the primary antibody: 1. We used a 1:500 dilution, much more concentration then recommended, yet did not produce a strong signal in the Western Blot. Our lab has had success with this protocol using other antibodies. 2. In repeat Westerns, the postive controls of both HSV1 and HSV2 are not being detected. 3. We have also tried using this Ab in immunofluorescence assays, none which have worked thus far.

I appreciate your time and concern with this inquiry, and I believe I supplied all the information I can. Most likely, we are as anxious to understand what is going on as you are, but I cannot invest any more time and due to these time constraints, we are pursuing ordering from another company.

Thank you,

ANSWER:

Thank you for providing these details. When this antibody was characterized in Western blotting with HSV-1 Antigen and the HSV-2 Infected Cell Extract, the antibody worked at dilutions from 1:10,000-1:80,000. As you're seeing a very weak signal using a dilution of 1:500, there definitely seems to be a problem with the antibody that you received.

I would be happy to provide you with a free of charge replacement or a refund or credit. Please let me know how you would like to proceed and I look forward to hearing from you.

Customer is using this antibody in WB with recombinant HSV2gd (strain 333) and after a one hour exposure saw a very faint band at approx 55 kDa. Primary was used at 1:500 and incubated for 1 hr at RT.

She also used HSV2 control (strain Hg52) and got no signal. Also used HSV1 - no band.

The antibody seems to be detecing recombinant protein, but not endongenous.

Lot# 142620

ANSWER:

Thank you for your enquiry. I was able to obtain the following information from our source for this antibody about how it was tested in Western blotting. Ab6507 was tested using the HSV-1 Antigen and the HSV-2 Infected Cell Extract at 10 micrograms/cm. A band at 55 kDa was detected, and you can see an image of this Western on the online product datasheet (I just added it). They used a general protocol, the only difference that I saw was that a BCIP/NBT detection kit was used. We always suggest using ECL+ as it's more sensitive.

Can you tell me more specifics about your samples and your Western protocol? It'll then be easier to see what may be going on with this antibody.

Thank you and I look forward to hearing from you.

We have recently purchased a batch of ab6507 antibody from Abcam (order number 111265). Despite the fact that this antibody used to work fairly well in ELISA in the past (please attached see our recent article where we cited Abcam), this new batch of the antibody is so weak in ELISA. We also compared one of the old batches (same product from Abcam) and the new one and it appeared that the new batch is much weaker than the old one. I would therefore request reimbursing us for this order.

ANSWER:

Thank you for your e-mail and congratulations on your paper.

I can certainly arrange a refund on your recent order of ab6507 if the antibody was purchased in the last 90days and if you could provide your order details. We have not received any other complaints about this antibody so it may have been damaged during shipping or storage; as you have tested the new lot with old lots in parallel this indicates that the rest of your experiment is working well and the antibody is damaged.

Can I please also suggest you submit an Abreview on the antibodies you have used and in return we will offer you 50 Abpoints per Abreview which can be redeemed on a number of rewards (a further 100 Abpoints will be offered for an image/figure).

I look forward to hearing from you regarding your order details,

Dear Technical expert:

I would like to do immunofluorescence using your mouse monoclonal to HSV gD --ab6507 (clone # 2C10).

Which fixatives are compatible with this antibody, and which of these is prefered? (i.e. 4% Paraformaldehyde in PHEM OR 100% Methanol OR %100 Acetone, OR 50:50 acetone methanol, etc).

Thanks for your response.

ANSWER:

I assume that you are doing frozen section work. 100% acetone for 10 minutes is usually a good fixative for this purpose. Aside, use PBS to dilute the antibody and for washes.