Anti-xCT antibody (ab37185)

Thanks so muchfor your reply and best of luck with your upcoming experiments. If you have any further questions please do not hesitate to contact me.

Thanks for submitting your enquiry. I am sorry to hear this antibody is giving you trouble.

I was just wondering what positive control samples you have used?

Thanks in advance for the additional information.

Thank you for contacting us and making us aware of this. We have been transitioning to a new email system and it may be that the response which I had sent on Mon 16 Apr 12 13:26GMT was somehow caught up or perhaps it went directly to your spam or junk mail boxes. Regardless prompt customer service is very important to us andI am sorry if it appears that your response was not sent in a timely manner to you at XXXX. If you could please send me the order number of the xCT antibody which you have purchased I would be happy to use that information to create a token of our appreciation. I have pasted the body the email response here: Thank you for your response and for the additional details which you have provided. You are correct as this antibody will only recognize the intracellular epitope of xCT. As such you would need to permeablize your cells to allow entry of the antibody. In the publication:Pampliega Oet al.Increased expression of cystine/glutamate antiporter in multiple sclerosis.J Neuroinflammation8:63 (2011).http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21639880&dopt=Abstractthey were able to use this product, conjugated to FITC with a concentration of 2.1ug/ml. Of course the appropriate concentrations would need to be determined experimentally in your protocol but this is an excellent starting point. At Abcam we do offer a number of different antibody conjugation kits for fast, easy and efficient direct conjugation of chromogen or enzymatic or biotin/streptavidin tag to your antibodies. We do recommend direct conjugation of the antibody in Flow cytometry as this eliminates the need for secondary antibodies and reduces complexity of the protocol. More information about our EasyLink direct conjugation kits may be found at the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=13148 Between April 1rst and May 31rst we are running a special promotion where you may receive a FREE primary antibody with purchase of an EasyLink conjugation kit. If you are interested in this offer please follow the link below: https://www.abcam.com/index.html?pageconfig=resource&rid=14962 Alternately, I would recommend using Ab111822 as this product has been produced from an extracellular region of xCT,amino acids 215-229. As this product has not been tested in Flow Cytometry however we cannot guarantee its effectiveness in that application. However we may offer to you our 100% Abreview testing discount should you wish to use this in Flow. With this program, you would purchase Ab111822, test in Flow Cytometry and leave an "Abreview". After the Abreview has been submitted, a code which I will provide to you will become active, entitling you to on e free primary antibody of your choice. If you are interested in participating in our 100% Abreview testing discount program please let me know and I will send further information to you. I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Thank you for your response and for the additional details which you have provided.

You are correct as this antibody will only recognize the intracellular epitope of xCT. As such you would need to permeablize your cells to allow entry of the antibody. In the publication:Pampliega Oet al.Increased expression of cystine/glutamate antiporter in multiple sclerosis.J Neuroinflammation8:63 (2011).http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21639880&dopt=Abstractthey were able to use this product, conjugated to FITC with a concentration of 2.1ug/ml. Of course the appropriate concentrations would need to be determined experimentally in your protocol but this is an excellent starting point.

At Abcam we do offer a number of different antibody conjugation kits for fast, easy and efficient direct conjugation of chromogen or enzymatic or biotin/streptavidin tag to your antibodies. We do recommend direct conjugation of the antibody in Flow cytometry as this eliminates the need for secondary antibodies and reduces complexity of the protocol. More information about our EasyLink direct conjugation kits may be found at the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13148

Between April 1rst and May 31rst we are running a special promotion where you may receive a FREE primary antibody with purchase of an EasyLink conjugation kit. If you are interested in this offer please follow the link below:

https://www.abcam.com/index.html?pageconfig=resource&rid=14962

Alternately, I would recommend using Ab111822 as this product has been produced from an extracellular region of xCT,amino acids 215-229. As this product has not been tested in Flow Cytometry however we cannot guarantee its effectiveness in that application. However we may offer to you our 100% Abreview testing discount should you wish to use this in Flow. With this program, you would purchase Ab111822, test in Flow Cytometry and leave an "Abreview". After the Abreview has been submitted, a code which I will provide to you will become active, entitling you to on e free primary antibody of your choice. If you are interested in participating in our 100% Abreview testing discount program please let me know and I will send further information to you.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Thank you for contacting Abcam, I am sorry to hear that you have been experiencing difficulties using this product in flow.

Would you be able to provide me the specific details of your experiments? Useful information would include but not be limited to:

Cell type, species and any treatments. Isotype controls used. Positive controls.
Have you used the secondary antibody with luck in flow?
Have you attempted to directly conjugate a tag to the antibody?
How many cells were used per experiment? What was the concentration of your lot and overall concentration of the antibody in your cell solution?
Have you attempted different concentrations?
Did you use a live/dead dye?
Any data is also very helpful.

I would also suggest reviewingPampliega O et al.Increased expression of cystine/glutamate antiporter in multiple sclerosis.J Neuroinflammation8:63 (2011). WB, Flow Cyt, IHC-FoFr; Human, Mouse, Rat.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21639880&dopt=Abstract, as they did extensive testing with this product in flow cytometry.

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, as you are using it according to specifications listed on our datasheet.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Thank you for contacting Abcam.

We are unable to send you any free/trail sizes of our antibodies. When your ordering system is reorganized at the end of the month, you will be able to order the antibody and if it is in stock then it will be shipped overnight for next day delivery. Therefore you will still be able to use the antibody on your fresh samples.

Please let me know if there is anything else I can help you with.

Thank you for your call today and for letting us know about the trouble with ab37185. As we discussed, please let me know if the suggestions that we discussed don't improve the results, and I will be happy to help you further. If you have any questions or if there is anything else that we can do for you, don't hesitate to let me know. Have a great weekend!

Thank you for your enquiry. As discussed on the phone, here is the email that was sent in response to your questionnaire from Kate Hayes on December 1st, 2006. Reply from Abcam to your enquiry regarding ab37185 Dear Ilangovan, In response to your enquiry (ref: 958900): BATCH NUMBER 227973 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE total protein from mouse brain PRIMARY ANTIBODY Abcam, mouse, two different concentrations, one at 0.5 ug/ml and 2.0 ug/ml was used. Incubated for over night at 4 deg C. Three times washed with TBS plus 0.1% tween 20 each for five minujtes DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED no positive and negative control was used ANTIBODY STORAGE CONDITIONS -20 deg Celsius SAMPLE PREPARATION tissue was homogenized in RIPA buffer supplemented with protease inhibitors AMOUNT OF PROTEIN LOADED 30 microgram ELECTROPHORESIS/GEL CONDITIONS Reducing, 10% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Transfer buffer 5.8 g Tris, 2.9 g Glycine, 0.37 g SDS and 200 ml methanol adjusted to 1 liter with deionized water, semi-dry transfer for one hour at 25 V, blocked with 5% non-fat dry milk solution SECONDARY ANTIBODY Invitrogen, goat-anti-rabbit, 1:2000, one hour, three times washed with TBS plus tween 20 each for five min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? none of the steps altered ADDITIONAL NOTES in xCT knock out samples, the protein is appearing with this antibody. We have an answer: Thank you for taking the time to complete our questionaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody (ab37185). I would like to offer some suggestions to optimise the results. I would also appreciate if you can confirm some details of the procedure. 1. In order to increase the signal,I can suggest increasing the antibody concentration. Dilutions on the datasheet are a guideline only. You can try 1:200 dilution. 2. Can you confirm you have reduced and denatured the samples in SDS and mercaptoethanol? This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. 3. Can you confirm if you have checked protein transfer onto the membrane with Poncea red? 4. How long was the blocking incubation? To ensure the membrane is not overblocked, I suggest this can be reduced. Try 1 hour, with just 3% blocking agent. BSA often gives better results than milk. 5. If you are obtaining faint bands at the correct size for xCT in the knockout samples, it could be that the knockout has not been efficient. Has this been checked, for example using PCR to detect if the xCT gene is still present? I would like to be able to suggest a negative control, but the xCT protein is ubiquitously expressed. I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again so I can investigate further. Best regards Kate Kate Hayes Technical Support Assistant Abcam plc https://www.abcam.com