Anti-EGFR antibody (ab2430)

Tried antibody in IHC-Fr sections mouse tissue kidney - instead of typical membrane pattern, only a nuclear stain was detected. This observation is in absolut contrast to all reported EGFR localization. Wondering whether the antibody works for this application (in contrast to your ABx specification on the data sheet). Any similar experiences by other customers? Thanks so much for your help - As we purchase a lot of ABx from ABCAM, and we had so far pretty good experience I´ll hope you can help us with this specific issue. Protocol: Cryosections -> PFA fixation 4% for 2 min Blocking with BSA5% and NDS 5% Primary antibody EGFR ab2430 1:100 in Blocking buffer for 1 h at RT Detection with appropiate anti-rabbit ALEXA 488 1:500 for 45 min Wash-steps 5 min 3 times after each incubation step Mounting with Prolong Gold antifade Image acquisition ZEISS confocal

ANSWER:

Thank you for contacting us. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Have you tried any other fixatives, such as acetone? PFA fixation may need antigen retrieval, so it is probably best to try with acetone as we've heard of this working well for other customers.

http://www.abcam.com/index.html?datasheet=2430&tab=abreviews&intabreviewid=18253

Have you run a no-primary control to ensure that the secondary isn't binding non-specifically?

Lastly, according to Swiss Prot, this protein, "In response to EGF, translocatedfrom the cell membrane to the nucleas via Golgi and ER." http://www.uniprot.org/uniprot/Q01279 Yes, this protein is most commonly found in the membrane, but could it be that you're doing some treatement or your samples are such that you are witnessing translocation? The listed subcellular location for mouse EGFR is: Cell membrane; Single-pass type I membrane protein. Endoplasmic reticulum membrane; Single-pass type I membrane protein. Golgi apparatus membrane; Single-pass type I membrane protein. Nucleus membrane; Single-pass type I membrane protein. Endosome. Endosome membrane.

Please let me know if the different fixation and no-primary control may be two things you can try. Other than that, the protocol looks fine. You can also try diluting the primary more, but I think the other two recommendations will help.

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.

Dear technical team,

Our customer would like toreceive CoC of ab2430 with lot#GR69660-1.
Please inform me for our customer.

Best regard,

ANSWER:

Thank you for contacting us and sorry for the delay in getting back to you.

Please find the Certificate of compliance attached as requested.

If you have any further questions please do not hesitate to contact us again.

Dear technical team,

Our customer would like to receive CoC of ab2430 (lot#GR69660-1).

Please send me the file.

ANSWER:

Thank you for contacting us.

I have attached the CoC for AB2430 as you have requested.

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

Thank you for your below reply and for your support.



Kindly find below in blue the customer’s reply to your inquiries concerning this antibody:



1. What is the lysis buffer that you have used? The lysis buffer used is laemmli buffer.

2. Have you reduced and denatured your samples? Which method/buffers did you use? The samples were denatured and reduced by heating at 95 degree Celsius for 10 minutes and then adding Beta-mercaptoethanol (10% of total volume).

3. How much total protein did you use per well? 25 microgrof each sample is added per well.

4. What membrane type did you transfer to? Nitrocellulose membrane is used for transfer (Amercham).

5. What blocking steps did you use? 5% milk (zero fat) is used for blocking (2.5 grams of this powdered milk is dissolved in 50 ml wash buffer: TBS tween 20).

6. What dilutions of this product did you use? How long and at what temperature was this antibody incubated? The primary antibody was used at 1/ 1000 dilution (for EGFR antibody ) shaking at room temperature for 2 hours.

7. What was the secondary antibody that you have used? How long and at what temperature was this antibody incubated? Anti-rabbit was used as secondary antibody. Dilution: 1/ 2000 at room temperature shakingfor 2 hours.

8. Have you run a no primary antibody control blot? What were the results? yes, theno primary antibody control shows no band.

9. What is the wash buffer that you use? TBS tween 20 is the wash buffer

ANSWER:

Thank you for providing that protocol information.

Having looked through the Abreviews that are on the website for ab2430, there are 2 protocol variations that I can suggest:

1 - Instead of using a 5% milk block, try using a 5%BSA block.

2 - Incubate the primary antibody in BSA, overnight at 4C, instead of at room temperature.

If these variations do prove to be successful, or if you feel that they will not work, then please let me know and I will be happy to either replace or refund the cost of the antibody. If you do wish to receive a replacement or a refund, then please provide me with the original Abcam order information and also the catalogue number that you would like to receive as a replacement.

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

We have received the following complaint from a customer who have previously ordered the “ab2430 Anti EGFR antibody”. In the datasheet of this antibody and in all the publications it is mentioned that this protein should appear at 170KDa, but in all his results obtained using different samples the protein appears at 70kDa and not 170kDa.   Kindly find attached to this message the data resulting from using this antibody sent from the customer, kindly advise?

ANSWER:

Thank you for bringing this to our attention. While EGFR has a number of isotypes that appear at 70kDA, this particular product should only recognize the 170kDA isoform. I would appreciate if the customer could provideparticulars of their protocol. This information is important not only in helping us understand the problems but in our internal testing as well.

As always, 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.

Below is a guide for the type of information which would be most valuable to me in understanding the procedure used:

Could you tell me the lysis buffer that you have used?

Have you reduced and denatured your samples? Which method/buffers did you use?

How much total protein did you use per well?

What membrane type did you transfer to?

What blocking steps did you use?

What dilutions of this product did you use? How long and at what temperature was this antibody incubated?

What was the secondary antibody that you have used? How long and at what temperature was this antibody incubated?

Have you run a no primary antibody control blot? What were the results?

What is the wash buffer that you use?

Could you provide the Abcam order confirmation number or PO number used to purchase this?

Thank you very much for taking the time to answer these questions. Please contact me if you have any questions or concerns.