Anti-HMGB1 antibody [HAP46.5] (ab12029)

Can I use ECL reagent as chromogen in immunohistochemistry.  

ANSWER:

Thank you for your reply. ECL is an appropriate reagent to use in Western blot, where the chemiluminescence can be developed on film. However, it can't be used in IHC. For IHC, we recommend using DAB or AEC substrate with an HRP-conjugated secondary antibody, or Fast Red substrate with an AP-conjugated secondary. We have DAB available as ab94665- http://www.abcam.com/DAB-substrate-kit-ab94665.html We also have AEC as ab64252- http://www.abcam.com/AEC-Substrate-System-Ready-to-Use-ab64252.html  And Fast Red as ab64254- http://www.abcam.com/Liquid-Fast-Red-Substrate-Kit-ab64254.html I hope this information will be useful, but if you have any further questions or need anything else, please let me know.

Hi

I brought primary antibody against HMGB1.

No. ab12029

Does it works for Immunohistochemistry? If so what is the dilution I have to use.

Thank you

ANSWER:

Thank you for contacting us with your question. We have only tested ab12029 in Western blot and ELISA, so it is possible that it will work in IHC, but we don't have any data in this application and we can not guarantee it. We have received some reports of background cytoplasmic staining in ICC (immunocytochemistry) on HeLa cells, so the protocol may need to be optimized if background staining is present.   For IHC using a purified antibody, we would recommend starting with 5 ug/mL, which would be ~1:400 dilution for ab12029. We also recommend blocking with 10% serum, and performing a heat-induced antigen retrieval with citrate buffer if the samples are paraffin-embedded and formalin-fixed. I hope this information is useful, but please let me know if you have any further questions and I'll be happy to help.

Has this antibody been epitope-mapped?

ANSWER:

Thank you for your inquiry.

I received confirmation that this antibody has not been epitope mapped.

I am sorry that this information couldn't be more helpful, but please let me know if you have any other questions.   

Our customer bought ab12029 (HMGB1) antibodies 17ea from last year. And she has been used this antibody for one year.

She believed this antibody was specific to detect HMGB1. But recently she bought HMGB1 knock out cell, and performed western blot using ab12029 and had a band although she expected no band. So she asked to you this antibody was specific for HMGB1 and you replied this ab12029 can detect HMGB2 also. In your datasheet there was no mention about the possibility of cross reactivity HMGB1 and 2, but other company specified the cross reactivity. (I attached the datasheet). And you also sell HMGB2 antibody separately. She believe this ab12029 is HMGB1,2 antibody not HBGB1. Her demands are below. 1. She has 4ea ab12029 so she wants to replace this to ab18256. When she tested ab18256 (HMGB2) there was no band in HMGB1 knock out cell. 2. She wants to see your test results that ab18256 is specific to detect HMGB1 not 2. I know you have no data, so she thinks Abcam have to test in abcam’s lab. She wants to confirm exact antibody’s specificity not possibility.   She is our very big customer, she use about fifty Abcam’s antibodies in one year from us.   I attached the data and wants to receive your prompt reply.   Best regards    

ANSWER:

Thank you for your reply.

I understand the customers concerns, and it is likely this antibody is detecting HMGB2.  We appreciate the results and feedback, and apologize for any inconvenience. I have updated the datasheet with this information.

In this case, I would be pleased to provide a credit note for these vials of  ab12029 the customer purchased. In order to arrange this, I would appreciate if you could please provide the Abcam order reference number and date of purchase for these vials.

I have been in contact with our source again, who is investigating this further. It is possible they may do some crossreactivity testing, but regrettably this is not always feasible. Any information we obtain we will use to update the datasheet.

Thank you for your cooperation. I look forward to hearing from you with the orders numbers and details of how you would like to proceed.  

Dear technical team,  

Our customer has tested ab12029 and ab67282 and their sizes are very close so,   would like to know the possibility they have cross reactivity between  HMGB I and HMGB II.  

Thank you.    

Best regards,

ANSWER:

Thank you for your enquiry.

The immunogen for ab67282 HMGB2 antibody has very low alignment to the HMGB1 protein, so is not likely to detect HMGB1.  However, this has not been specifically tested in the laboratory.

The ab12029 HMGB1 immunogen does have higher alignment to the HMGB2 protein, and so this antibody may also detect HMGB2, but I am sorry we do not have any experimental data to confirm this.

I hope this information will be helpful to you. If you have any further questions please do not hesitate to contact us.