Anti-Hepatitis B Virus Core Antigen antibody [C1] (ab8637)

Thank you for contacting Abcam regarding ab8637.


I am sorry that you are experiencing difficulties with this antibody in WB. In order to assist you further, I wanted to clarify several points in your protocol.


You mention that you load 15ul of protein - do you know what the quantity of protein is in ug? Was the protein isolated from a particular cell type (tranfected)or translated in vitro (purified protein)? What is the difference between each of the three samples?


Here are the recommended WB protocol conditions:


Following lysis, prepare samples in Laemmli buffer and boil 10 minutes prior to loading.
Load 50ng of purified protein or 50 - 100ug cell lysates
Prior to WB, confirm efficient transfer to the membranes by Ponceau S

WB conditions:
Block - 5% milk (or preferrably BSA) in TBSTfor 1 hr at RT
Primary antibody - dilute ab8637 1:1000 - 1:5000 in 5% milk (or BSA) and incubate o/n at 4oC or 1 hr at room temperature
Wash 3X 10 min in TBST
Secondary antibody (HRP conjugated)- dilute according to manufacturers instructions and incubate 1 hr at RT
Wash 1X 15 min and 3X 10 min in TBST

Develop using ECL



Based on the results you provided, you are getting the band of interest with some background that can be cleaned up by modifying the protocol a bit. I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

Thank you for your reply.

Upon further investigation, this target contains several phosphorylation sites which may account for the shift in molecular weight. Otherwise, I cannot account for the shift.

If you would like to try another vial of this antibody, I would be happy to send you one. Please let me know if you are interested.

I look forward to your reply so that I may assist you further. Please do not hesitate to contact us if you have any additional questions.

Thank you for contacting Abcam regarding ab8637. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information and image you provided and would like to ask some additional questions. There seems to be a decrease in the expression of your protein of interest in the lanes from left to right. Were these treated differently in any way? GAPDH appears consistent, but I am not sure if you have treated the cells. What percentage gel did you run? Was everything run under reduced, denatured conditions? How long did you boil the samples before loading? It is recommended to boil the samples for at least 10 minutes for this antibody to work well. Also depending on the percentage gel and sample preparation, the molecular weight may run slightly different than the predicted molecular weight. I hope these guidelines are helpful. I look forward to your reply so that I may assist you further. Please do not hesitate to contact us if you have any additional questions.

Thank you for your question. I can confirm that this Hepatitis B Virus Core Antigen antibody, ab8637, will recognize native core particles but not native e antigen. Under denaturing conditions, it willr ecognize all core polypeptides, containing epitope around aa 80 (core, precore, e antigen etc.). Differentiation of core and e antigens in liver tissues using this antibody, and several others, has been published in: J Med Virol. 1997 Oct;53(2):127-38. Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies.Naoumov NV, Antonov KA, Miska S, Bichko V, Williams R, Will H. This antibody will recognize all core polypeptides (core, precore, e antigen etc.) under standard western blot conditions. It appears that at least partial renaturing of proteins occurs on the membrane, so no additional renaturing steps are necessary. I hope this information is helpful. Should you have any further questions, please do not hesiate to contact us again.

Ab8637 and ab8639 should recognize the precorerotein in WB but there is no "cross-reaction" as all proteins are distinguishable by mobility on the gel (i.e they will recognise both the precoreprotein and core protein). AB8638 may or may not recognize your protein fragment aa1-150, since its epitope is aa aa35-140 of the core protein, which is really close to the N terminus of the protein you are going to analyze. Ab8637 will not recognize the precorprotein under native conditions, because this protein can not self assemble into particles. I hope this information will help you, please let me know if you need further assistance,

Here is the information that I could obtain regarding ab8639, ab8638, and ab8637. The recommended starting dilutions are 1:1,000 and down in 10-fold increments. As for detection, any known detection method would work. Once again, those mabs had being characterized in great detail in the following paper: Bichko et al. Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Mol Immunol. 1993 Feb;30(3):221-31 If you have any more questions, just let me know.

I have received the following information regarding ab8255 and ab2045, but will have to get back to you about the other antibodies. The originator has said that they tested the antibodies as pairs in internal ELISA. Possible pairs are H6F5 (ab2045) - H3A4 (ab8255), and H3A4 - H6F5. They have used coating concentration 5 - 10 ug/ml and conjugate concentration 0.2 -1 ug/ml. However, the concentrations need to be optimized separately for every application.

Thank you for your enquiry. I was able to obtain the following information regarding the use of these antibodies in ELISA. Ab8255 and Ab2045: These antibodies have been tested in ELISA with the native antigen. The antibodies can be used as a pair in sandwich ELISA. Both clones can work as capture and as detection antibodies. Ab8638, Ab8637, and Ab8639: These antibodies work well with a recombinant or natural HBcAg adsorbed on a plate, or in sandwich ELISA. Also, they work well with the synthetic HBcAg peptides. Below is one reference: Bichko et al. Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Mol Immunol. 1993 Feb;30(3):221-31

Thank you for your reply. The only data we have regarding this is that it is an ayw variant as described in: Bichko et. al. Subtype ayw variant of hepatitis B virus. DNA primary structure analysis. FEBS Lett. 1985 Jun 3;185(1):208-12.