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Reassurance, Refunds & Replacements
If your product does not perform as described on this datasheet, we will refund or replace your product...
Read our guarantee »This product is covered by the Abpromise guarantee. Our scientific support team are available to answer any questions or queries - fill out an inquiry form for ab30 for help.
Alternatively, you can search the previous enquiries about this product to see if your query has already been answered.
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phone call: searching for monoclonal antibody for flow cytometry on rat cells |
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ANSWER: |
Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten. |
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Thank you very much for giving me many advice. |
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ANSWER: |
Thank you for your reply. |
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Would like to stain EGFR of live H1703 cells. |
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ANSWER: |
Thank you for your call today and for our discussion of these EGFR antibodies. |
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BATCH NUMBER 254 ORDER NUMBER replacem DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Recombinant human sEGFR-extracellular http://www.researchd.com/cytokines/recpt.htm PRIMARY ANTIBODY Ab30 / diluted by PBS / 1?F1000 / room temperature for 1 hour / PBST wash 3 times SECONDARY ANTIBODY PIERCE 31320 / goat / PBS / 1?F5000 / room temperature for 1 hour / PBST wash 3 times DETECTION METHOD NEOGEN #304177 K-Blue® MAX Substrate (TMB) POSITIVE AND NEGATIVE CONTROLS USED the sample is a postive control ANTIBODY STORAGE CONDITIONS 10 ul aliquots in - 20 ?? TYPE OF ELISA Direct ELISA COATING WELL PBS 100 ng / well BLOCKING CONDITIONS StartingBlock Blocking Buffer / room temperature for 1 hour http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=65B71909-7ABF-4D52-8401-3107D9977640 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? none ADDITIONAL NOTES This lot is an replacement. (Enquiry detail for enquiry ID: 358940). Our customer repeated the ELISA assay three times but the signal remains to be very weak.(Raw data is available) Our customer has generated EGFR antibody which works well in ELISA and WB and needed a commercial antibody (ab30)to serve as a positive control. But it seems like the titer of ab30 is extremely low. Our customer would like to try ab231 if a replacement can be processed again for this case.
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ANSWER: |
I'm sorry to hear you are still having problems with the replacement of ab30, this must be very fustrating. I understand that my colleague Danielle has raised the possibility that "this antibody does not recognise recombinant EGFR. The epitope has been mapped to between aa 6-273 of human EGFR and this stretch of AAs is included in the recombinant protein used in this research. The epitope maps to the external EGF-binding domain of the EGFR transmembrane glycoprotein and perhaps this region of the recominant protein is not in the correct configuration for the antibody to bind" (e-mail from 28th Sept 2004). I would like to suggest trying an overnight incubation with the primary antibody (at 4C) to enable better binding and if this is still unsuccessful we can arrange for a credit note or replacement with ab231 (please note this antibody had not been tested for ELISA) . Please let me know if this helps and do not hesitate to contact us for further advice, |
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BATCH NUMBER 254 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal SAMPLE 50 ng rhEGFR protein?a.a. 25 - 645? R&D cat#1095-ER PRIMARY ANTIBODY Abcam 30 Diluent:TBSTx3 (5 min per wash) Dilution:1:1000 Time:1hr Temp:RT SECONDARY ANTIBODY PIERCE #31430 ImmunoPure Goat Anti-Mouse IgG (H+L), Peroxidase Conjugated TBSTx3 1?F5000 1hr at RT DETECTION METHOD NEOGEN #304177 K-Blue® MAX Substrate (TMB) POSITIVE AND NEGATIVE CONTROLS USED Positive control=our sample ANTIBODY STORAGE CONDITIONS -20 degree C, ddwater TYPE OF ELISA Direct ELISA COATING WELL 1xPBS 50ul of sample 50ng protein per well BLOCKING CONDITIONS Pierce cat#37542 Starting Block blocking buffer, 1hr RT 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? No
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ANSWER: |
The protocol that your customer is using looks fine so I would be happy to send you a replacement vial just in case there is a problem with the original vial that they received. However, it is possible that this antibody does not recognise recombinant EGFR. The epitope has been mapped to between aa 6-273 of human EGFR and this stretch of AAs is included in the recombinant protein used in this research. The epitope maps to the external EGF-binding domain of the EGFR transmembrane glycoprotein and perhaps this region of the recominant protein is not in the correct configuration for the antibody to bind. Please let me know if you would like me to include another vial on your next order. |
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
](/ps/datasheet/images/0/ab30/EGFR-Primary-antibodies-ab30-1.jpg)
![Flow Cytometry - EGFR antibody [EGFR1] (ab30)](/ps/datasheet/images/0/ab30/EGFR-Primary-antibodies-ab30-2.jpg)
![EGFR antibody [EGFR1] for Immunoprecipitation in Mouse (30)](/ps/datasheet/images/0/ab30/EGFR-Primary-antibodies-ab30-4.jpg)