Anti-ErbB 3 antibody [RTJ2] (ab20161)

Hello Tech Team, I Have a customer that would like to know the concentration of the current lot AB20161- Anti-ErbB 3 antibody [RTJ2] Thank you for your help and hope to hear from you soon.

ANSWER:

Thank you for contacting us.

We currently have two lots in stock with different concentrations:

lot number GR51890-1 / 1133680     1.36 mg/ml lot number GR37977-1 / 1074564     1.27 mg/ml (very few units available)

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

I didn’t realize that the ErbB3 antibody (ab20161) was directed against the cytoplasmic region of ErbB3.  That should explain why I didn’t observe any signal during FACS analysis after I observed the ErbB3 signal via Western blot in whole cell lysate using the same antibody. 

What method would you recommend I use to fix and permeabilize the cells.

Several methods for intracellular staining are listed on your website, but I noticed that under ‘special recommendations’ it states that “Antigens

close to the plasma membrane and soluble cytoplasmic antigens will require mild cell permeabilisation without fixation”.  Should I not fix my cells then?  Your insight into this issue would be greatly appreciated. 

Thank you again for your time.

ANSWER:

Thank you for your reply.

I would suggest to look at the overlay histogram shown on the product datasheet. HeLa cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab20161, 1µg/1x106 cells) for 30 min at 22ºC.

Alternatively, we also recommend to fix the cells with 0.01% formaldehyde for 15 min at 4°C. The permeabilisation can be done using a detergent such as 0.5% Saponin (a mild membrane solubiliser).

I hope this is helpful. Do not hesitate to contact us again if you have further questions.

I recently purchased your mouse monoclonal [RTJ2] to ErbB3 antibody (ab20161) to analyze expression of ErbB3 on the cell surface of BT-474 (human) cells via flow cytometry.  I have previously tested this cell line via FACS analysis using a different antibody acquired from BioLegend and was able to obtain good results (This anti-human ErbB3 antibody has PE directly linked to it and therefore requires no secondary antibody for flow Cytometry analysis).  Unfortunately, the BioLegend antibody previously used does not work for the Western blot assay.  The product information sheet for ab20161 claims that it is good for flow cytometry and Western blots.  However, when I used your antibody at 2 ug per 1 million cells and using your goat F(ab’)2 polyclonal

secondary antibody to mouse IgG – H&L (PE) pre-adsorbed (ab98732) @ a 1/50 dilution, I did not observe any PE signal over background levels.  Is there another secondary antibody that you would recommend to use in conjunction with ab20161?

Thank you for your time.

ANSWER:

Thank you for contacting us. I am sorry to hear that these antibodies are not providing satisfactory results.

Having reviewed this case, . I would also appreciate if you can confirm some further details:

- is the primary ErbB3 antibody ab20161 working fine in Western Blot?

- the primary ErbB3 antibody ab20161 is directed against the cytoplasmic region of ErbB3. How did you fix the cells? Did you permeabilise the cells before staining?

- was the secondary antibody ab98732 stored in the dark at 4°C?

Since at Abcam we are concern about the quality of the products we supply, I would like to ask you further information regarding the samples you tested and the protocol you used. Could you please fill in the attached questionnaire? We will use those data to investigate the source of the problem with this product.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Which region of the protein does this antibody recognize: extracellular or cytoplasmic?

ANSWER:

Thank you for your enquiry.

The peptide used to generate ab20161 is from a cytoplasmic region of the protein, amino acids 1004 - 1016. The peptide sequence has been placed on the datasheet.

Please do not hesitate to contact us if you have any other questions.