Anti-BCRP/ABCG2 antibody [BXP-53] (ab24115)

Thank you for getting back to me. This customer is very appreciated for your help. Regarding the EMSA result, she would like further to know whether the result was good or bad. Would you please help provide some comment or other information?

ANSWER:

Thank you for contacting us.


I have discussed the EMSA results with my colleagues, and to be honest we do not have any specificsuggestions:

Because there are many unique requirements for each nucleic acid binding protein and the corresponding antibody, there is no universal set of reaction conditions. If no information on prior work is available, conditions should be determined empirically.In general, the supershift assay is a step which can be optimised in various ways. So could the incubation time and temperature be altered and may range from for example 1 hour to overnight at 4C or 15-45 min at room temperature. Or the antibody could be addedin different concentrations before or after the oligos. Also, factors that affect the strength and specificity of the protein:DNA interactions include the ionic strength and pH of the binding buffer, the presence of nonionic detergents, glycerol or carrier proteins (e.g., BSA),the concentration and type of competitor DNA present.

If the customer has tried various conditions (which I am sure with her experience she has) and provided the ABCG2 protein construct is capable of binding to DNA, it might well be that the antibody is not suitable for EMSA. Antibodies being used to create a supershift may not work in a binding buffer that is optimal for a protein:DNA shift complex.Many nuclear proteins function well for shift complexes under reducing conditions. Therefore concentrations of DTT at 1 mM and higher are often added to the EMSA binding buffer. Yet, many antibodies become reduced and loose functionality in reducing conditions. Also, the epitope may not be in an appropriate location which preventsbinding of the antibody when the protein is bound to DNA.

Therefore,the outcomeof theexperiments seems unfortunately negative, however, it is very good toknowunder which conditions the antibodydoes not work!

Thank you very much for your time and contribution! I am sorry that I cannot be of more assistance on this occasion to help improve the results.

Do you offer a cold epitope for ab24115 BXP-53 Rat monoclonal antibody to BCRP/ABCG2? I am looking to use a cold epitope for blocking prior to primary antibody incubation during for immunoblotting. Thank you for your interest in this matter.

ANSWER:

Thank you for contacting Abcam. Unfortunately we do not have a blocking peptide available for this product. The sequence that was used to generate this antibody was ******** of Mouse BCRP/ABCG2 (SwissProt: Q7TMS5). You could have a peptide synthesized against this sequence and use that peptide for your blocking experiment. If there is anything else I can help you with, please let me know.

Thank you for your kind reply. :) This customer would like to apply the Discount code and test ab24115 in EMSA (human sample). Would you please help this customer create the Discount code?  

ANSWER:

DISCOUNT CODE: xxx Expiration date:dd mm 2012 

I am very pleased to hear you would like to accept our offer and test ab24115 in EMSA. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for EMSA and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative.

If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Our customer raise an inquiry about the Anti-BCRP/ABCG2 antibody. She is preparing the reagent for EMSA, which she thinks it may need much higher concentration of the primary antibody. Therefore, she would like to know whether ABCAM provides the antibody-concentrated service of our antibodies. That is, could ABCAM offers the higher concentration than the originally supplied product?

ANSWER:

Thank you for contacting us.

Unfortunately, we do not offer other concentrations of the antibody than the 0.25 mg/ml provided. However, we have antibody concentration kits available for the customer to concentrate ab24115. These are: ab102776: Click here (or use the following: http://www.abcam.com/index.html?datasheet=102776). ab102778: Click here (or use the following: http://www.abcam.com/index.html?datasheet=102778).

To our knowledge, ab24115 has not been tested in EMSA yet. Therefore, I can offer a discount off a future purchase if you buy ab24115 now, test it in EMSA and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody. If you are interested in this offer, please follow these steps: 1. Reply to this e-mail to let me know that you would like to proceed and test ab24115 in EMSA. I will then send a discount code. This code must be issued before purchasing ab24115 so please wait for my reply before ordering. 2. Purchase ab24115 either by phone, fax, or online (www.abcam.com). 3. Test it in EMSA 4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out more about our Abreview system, please visit: http://www.abcam.com/abreviews. 5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code.

The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue. We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab24115 turns out to be unsuitable for EMSA, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further. The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.  

Hello,

I want to ask, if you could recommend a positive control when using the ABCG2-Antibody for flow cytometry? Maybe bone marrow works as a positive control, but there are less than 1% of cells positive for ABCG2? Do you know, if there is a suitable cell-line as a positive control?

Thank you and regards

ANSWER:

Thank you for your enquiry.

I can confirm bone marrow or kidney tissue will be suitable as a ABCG2 positive control. Several cancer cells lines have been subcloned and selected for overexpression of ABCG2 protein. I can suggest MCF-7/MR and MCF-7/FLV1000 cells would be suitable (from the following reference):

Cancer Research 65, 10952-10958, December 1, 2005, Novel Extracellular Vesicles Mediate an ABCG2-Dependent Anticancer Drug Sequestration and Resistance. Ilan Ifergan1 et al.

I hope this information is helpful. Should you require any further information please do not hesitate to contact us again.