Anti-xCT antibody (ab37185)

Hi There,

 

I wrote you emails to explain my concern of using your xCT antibody to do flow cytometry and ask for your help. But I did not receive any response after I provided you with my experiment details, which makes me very upset. My lab ordered this specific antibody from your company at least 4 times, I think I deserve better service!

 

Below is the past correspondence with you. 

 

On Apr 13, 2012, at 10:16 PM, Yuzhe Yang wrote:
Hi Keith, 
I used human breast cancer cell line MCF-7 cells. Negative control: cells only, secondary anti rabbit IgG fitc only. No positive control. I used up the antibody for real experiments..
FITC secondary antibody was newly ordered, it is working very well in our lab's experiments.
I did not try to directly conjugated a tag to the antibody. How to do that?
100000 cells per sample in 500ul flow buffer (BSA + PBS + N3NA). I put 10ul of you shipped antibody per test. I tried 5 ul to 10 ul of the antibody. Never works. (what's your recommended concentration? I did not find the information in the data sheet..)
My cells should be live cells. I did not use live/dead dye.
 
Your antibody targets the N terminal of xCT protein. I searched xCT's structure, N terminal locates intracellularly. Does that mean I could not use the antibody to target cell surface expression of xCT protein? It seems only your company carry xCT ab for flow. I am quite desperate to get it to work! Thanks!
 
Mindy
 

On Apr 13, 2012, at 6:15 PM, mailto:technical@abcam.com wrote:



http://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header

Dear Sir/Madam
 
 
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 reviewing Pampliega O  et al. Increased expression of cystine/glutamate antiporter in multiple sclerosis. J Neuroinflammation 8:63 (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.

Help us improve our service. 
http://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3683952
Best regards, 
Keith 

Keith Beadle 
Scientific Support Specialist 
Abcam Inc. 
http://www.abcam.com/

Your original inquiry to Abcam:

Hi There,

Our lab recently purchased xCT antibody ab37185 for flow cytometry study. I want to measure xCT cell surface expression. So I first incubated my cells with ab37185 (10ul per sample) for 1hr and washed for 2˜3 times with PBS then incubated the cells with anti-rabbit IgG FITC antibody. However, I could never get a signal by flow. I am wondering what's wrong. Can you give me some suggestions? Thanks!



 
 


Abcam Customer Services and Scientific Support Team
http://www.abcam.com/index.html?pageconfig=technical&utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Body
[CCE3683952]



Discover more at http://abcam.com/

ANSWER:

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:

http://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:

http://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.

Hi Keith,

I used human breast cancer cell line MCF-7 cells. Negative control: cells only, secondary anti rabbit IgG fitc only. No positive control. I used up the antibody for real experiments..

FITC secondary antibody was newly ordered, it is working very well in our lab's experiments.

I did not try to directly conjugated a tag to the antibody. How to do that?

100000 cells per sample in 500ul flow buffer (BSA + PBS + N3NA). I put 10ul of you shipped antibody per test. I tried 5 ul to 10 ul of the antibody. Never works. (what's your recommended concentration? I did not find the information in the data sheet..)

My cells should be live cells. I did not use live/dead dye.

Your antibody targets the N terminal of xCT protein. I searched xCT's structure, N terminal locates intracellularly. Does that mean I could not use the antibody to target cell surface expression of xCT protein? It seems only your company carry xCT ab for flow. I am quite desperate to get it to work! Thanks!

ANSWER:

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:

http://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:

http://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.

Hi There,

Our lab recently purchased xCT antibody ab37185 for flow cytometry study. I want to measure xCT cell surface expression. So I first incubated my cells with ab37185 (10ul per sample) for 1hr and washed for 2˜3 times with PBS then incubated the cells with anti-rabbit IgG FITC antibody. However, I could never get a signal by flow. I am wondering what's wrong. Can you give me some suggestions? Thanks!

ANSWER:

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.

I'm wondering if you could send me a aliquot of your xCT antibody ab37185.
Indeed, I would like to test it on fresh samplesthat will beready at the end of the monthand, at the moment, we are not able to make any order (indeed we are changing oursystemof prurchase).At the end of the month however, everything will be OK and we will be able to order again. I could then regularize the order if you are agree.
Let me know your decision, hoping you will be able to send mean aliquotas soon as possible.

ANSWER:

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.

Multiple bands in addition to specific band,using human primary blood samples.

ANSWER:

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!