Anti-Coxsackie Adenovirus Receptor antibody [E1-1] (ab9891)

LOT NUMBER -- NOT SPECIFIED --
ORDER NUMBER -- NOT SPECIFIED --
DESCRIPTION OF THE PROBLEM No staining
SAMPLE Human lung sections from parrafin embedded tissue
PRIMARY ANTIBODY ab9891 diluted 1/10 (following recommended dilution for frozen sections) Incubated 1 hour at room temp
DETECTION METHOD DAB POSITIVE AND NEGATIVE CONTROLS USED We are trying to optimise this to use as a positive control Using Mouse IgG as a negative control
ANTIBODY STORAGE CONDITIONS -20'C Aliquoted to prevent repeated freeze/thawing
FIXATION OF SAMPLE Don't know
ANTIGEN RETRIEVAL Heat mediated in citrate buffer pH6 or TrisEDTA buffer pH9 Or Proteinase K
BLOCKING CONDITIONS Rabbit serum 1 hour at room temp
SECONDARY ANTIBODY Biotinylated rabbit anti mouse polyclonal. Using standard 1/200 dilution that successfully detects other primary antibodies. Incubated 30 mins at room temperature
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3
HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? I have tested different methods for antigen retrieval (heating in Citrate buffer pH6, or TrisEDTA buffer pH9 or proteinase K treatment.
ADDITIONAL NOTES What antigen retrieval method is recommended for use with this antibody? What antibody dilution is recommended for IHC in parrafin embedded samples

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

In the event that a product is not functioning in the applications cited on the product data sheet within our guarantee period of 6 months, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab9891. I would also appreciate if you can confirm some further details:

1. Please confirm the order number and date of purchase? I'm sorry I cannot trace any order for this antibody from Glasgow in the last 6 months. We do have an order placed 2nd March 2010, PO 1170287. Is this the correct order?

2. I am sorry to confirm this is not tested and guaranteed for IHC-P, and so we do not have information to provide regarding recommended dilutions of antigen retrieval that have been used previously. All tested and guaranteed species are listed on the product datasheets. I can suggest it would be important to consider to try a frozen section as this has been tested.

3. As far as I am aware, Coxsackie Adenovirus Receptor is expressed at a lower levels in the lung. I can suggest it would be important to consider including a positive control sample with higher expression level, for example pancreas, brain or heart tissue.

4. It would be important to find out about the fixation if possible. Is there any further information?

5. For antigen retrieval, this can often require some optimization. I can recommend to try different time points, for example 3, 5, 10 and 20 minutes if this has not already been tried.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

The positive control listed for this ab is HEK293 cells. Do these cells express the protein endogenously? Do they express it strongly? Do you have a WB image?

ANSWER:

Thank you for your inquiry.

I heard back from the testing laboratory concerning HEK293, and they only have data with FACS, but not Western. As HEK293 are positive for FACS and very sensitive for species C adenovirus infection, I would assume they also should express large amounts of CAR protein detectable in Western. Importantly as mentioned in the application notes, Western signals are much stronger when samples are treated under non reducing conditions, meaning the sample buffer should not contain DTT or mercaptoethanol.

I hope this information helps. Please contact us with any other questions.

I am interested in working with your antibody, mouse monoclonal[E1-1] to Coxsackie Adenovirus Recepter. I have already used this antibody to demonstrate CAR expression of lymphocytes. This datasheet said that “Use 50ul of antibody and a 100ul suspension of maximally 1 million cells.” Does this mean that there is nonspecific binding by your antibody fashion if used in excess of this amount? When I used 50ul it did not work but I used 100ul it did work. Does this mean that the activity of the antibody is weak? I would be very happy if you could tell me about it.

ANSWER:

Thank you for your enquiry.

As experiments may differ from one to the other, we strongly suggest that optimum dilutions and concentrations be determined by the end user. Please bear in mind that although the datasheet for this product ab9891 states “Use 50ul of antibody and a 100ul suspension of maximally 1 million cells.” , this is only a recommended starting dilution. It is always best to start with a titration (25ul, 50ul, 100ul)experiment to determine the best concentration/dilution to use. Anyway, I am happy to hear that you manage to find your optimum condition when using 100ul of suspension.

I hope my explanation helps resolve your worries about the product being non-specific or weak. If you have any other questions please do not hesitate to contact me.

What are the Western blotting conditions to be used with this antibody?

ANSWER:

Thank you for your enquiry. To use this antibody in Western blotting, please use non-reducing conditions. Omit beta-mercaptoethanol and DTT from the loading buffer but leave the SDS. Also, the migration buffer should contain SDS. Other than that, you can follow a standard Western protocol.

If you have any additional questions, please contact us again. Have a good weekend!

I am interested in purchasing your Coxsackie adenovirus receptor antibody (ab9891) for WB application. Could you tell me why the PAGE should be done under non reducing conditions?

ANSWER:

Thank you for your enquiry. When protein samples are reduced, Western staining is much weaker. The antibody can not bind efficiently to the reduced form of the protein, probably because the epitope consists of a nonlinear structural motif that is dependent on a disulfide bond. I hope that this helps, best of luck with your research.