Anti-MUC1 antibody (ab15481)

Dear Abcam, i would like to perform an ELISA sandwich assay using this antibodies:

1) Anti-MUC1 antibody C595 NCRC48, Mouse monoclonal, (ab28081) 2) Anti-MUC1 antibody, Rabbit polyclonal, (ab15481)

i've to question:

1) Can these antibodies use together? 2) Can you suggest me a compatible MUC1 protein? with my best regards,

ANSWER:

Sorry for the delay in contacting you with regards to this query. We have a number of pairs of mouse monoclonals which can be used to detect the under or unglycosylated form of MUC1 (for example ab10117 which can be paired with HRP conjugated ab24471). However if you are interested in the glycosylated form of MUC1, unfortunately we have not tested any antibody pairs in order to detect this form and therefore cannot guarantee that it will work. However, form the pair which you presented, ab28081 has been tested in ELISA with human protein and should therefore be suitable for this application. However, I cannot be sure that the rabbit polyclonal ab15481 will work as it has not been tested in ELISA before and has only been used in applications where the immunogen may be in the denatured form. Two possible alternative would be ab79226 which recognises the Y1229 phosphorylated form (cytoplasmic) or ab14690 which again recognises the underglycosylated form (extracellular) but have both been tested using ELISA. Alternatively, ab80952 is an Armenian Hamster monoclonal reactive against the cytoplasmic tail of MUC1, or ab37435 which is a rabbit polyclonal, again directed against the cytoplasmic domain. Alternatively ab45167 is a rabbit monoclonal which recognises the extracellular domain but only of isoform 7 or MUC1. These have been used in flow cytometry and immunoprecipitation and are therefore more likely to be able to detect the native form of the protein. If you would like to test ab15481 as you suggested (or any of the other antibodies suggested which have not been used in ELISA yet), as it has not been used in ELISA before I can offer a testing discount. This would involve you purchasing the antibody, using it and letting us know the results via an Abreview (positive or negative), you would then be eligible for a free primary antibody of your choice from our catalog. More information on this offer can be found here: www.abcam.com/collaborationdiscount If you need a secondary antibody for this application we have a very easy to use search engine to allow you to find exactly what you are looking for: http://www.abcam.com/index.html?pageconfig=productmap&cl=918 If you would like any further information, help in choosing a suitable secondary antibody or you are interested in applying for the testing discount please do not hesitate to contact me.

I found out that you sell a large number of antibodies against the human cell surface antigen MUC1. Does any of the ones you sell react with the extracellular domain further out than the cleavage site (or with the cleavage site)? Also, do they react with the mature form (several of the muc1 abs only react with low glycosylated precursors)? And thirdly, do they react with rhesus monkey MUC1?

ANSWER:

Thank you for your enquiry regarding our MUC1 antibodies.

There is indeed a large selection of those antibodies available in our catalogue and I have summarised below the information we have regarding the epitope recognised and whether we know if the antibody recognises various glycosylated forms of the protein. The extracellular part of the human protein is located with aa24-1158. Many antibodies recognize The APDTR repeat found in this region as you will see below. None of those antibodies have been tested against Macaca mulatta tissue, and a homology search showed 78% homology between the human protein and the monkey protein which may be low for using monoclonal antibodies.

Ab8323- Binds with high efficiency to epitope within the VNTR tandem repeat peptide region of MUC1 molecule. Recognizes underglycosylated and natural MUC1 protein.

Ab696. epitope not mapped. This antibody is specific to 300 kD DF3 antigen.

Ab14690. Immunogen sequence is unknown (the antibody is not made by Abcam and kept confidential from us). It is a “60 aa long synthetic peptide representing a 20 amino acid repeat sequence from mucin 1". The antibody labels underglycosylated mucin.

15481. Immunogen sequence is unknown (the antibody is not made by Abcam and kept confidential from us). We know the immunogen is from the cytoplasmic tail part of the protein. Type of underglycosylated or mature protein recognized: unknown.

Ab8665: It recognizes the multiple protein epitope that is relatively insensitive to glycosylation. Therefore, it is very sensitive in the detection of episialin expression in different epithelial tissues. Multiple epitopes.

Ab22711. SM3 antibody recognises the under-glycosylated form of MUC1. Epitope not mapped.

Ab8605. epitope not mapped. Type of underglycosylated or mature protein recognized: unknown.

Ab8618. The dominant epitope of this antibody is the 5-mer PDTRP of MUC1 tandem repeat as established with “epitope fingerprinting.” Type of underglycosylated or mature protein recognized: unknown.

Ab8606: The dominant epitope of this antibody is the 12-mer GVTSAPDTRPAP of the MUC1 tandem repeat as established with “epitope fingerprinting”. Type of underglycosylated or mature protein recognized: unknown.

Ab8608. The dominant epitope of this antibody is the 7mer TSAPDTR of the MUC1 tandem repeat as established with epitope fingerprinting. Type of underglycosylated or mature protein recognized: unknown.

Ab8607: The dominant epitope of this antibody is APDTR as established with “epitope fingerprinting.” Type of underglycosylated or mature protein recognized: unknown.

Ab8322, ab10115, ab22754, ab22751. Mabs bind with high efficiency with different regions of VTSAPDTRPAPGSTAPPAHGVTSA synthetic peptide spanning the one repeat of VNTR extracellular portion of MUC1 molecule. They react with VNTR5 and VNTR20 recombinant unglycosylated fragments of MUC1 protein and underglycosylated MUC1 prepared from tumor fluids or as a result of chemical treatment of human milk MUC1. These Mabs do not recognize natural MUC1 protein isolated from human milk by affinity purification on carbohydrate epitope specific Mabs.

Ab10118, ab10119 Mabs bind with high efficiency with different epitopes within the VNTR tandem repeat peptide region of MUC1 molecule, which have different conformations and peptide- carbohydrate compositions. Ab efficiently recognize underglycosylated and natural MUC1 protein isolated from human milk by affinity chromatography.

Ab10120. Mabs bind with high efficiency with different epitopes within the VNTR tandem repeat peptide region of MUC1 molecule, which have different conformations and peptide- carbohydrate compositions. Mab M4H2 reacts with VNTR20 recombinant unglycosylated fragment of MUC1 protein (and very weakly with monomeric MUC1 peptide), with deglycosylated and underglycosylated natural MUC1 (but not with completely glycosylated natural milk MUC1).

Ab10124. epitope: peptide repeat RPAP region. Type of underglycosylated or mature protein recognized: unknown.

Ab10114. epitope: Peptide tandem repeat in MUC1 core. Type of underglycosylated or mature protein recognized: unknown. Ab6261, ab853. EPitope: unknow. This antibody reacts with an antigen of 265-400 kD belonging to a heterogeneous group of heavily glycosylated proteins called human milk fat globule proteins.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information to help you choose the correct antibody suitable for your needs.

Can you please tell me the BSA concentration in this item?

ANSWER:

The BSA concentration is 0.2mg/ml.

BATCH NUMBER 105287 ORDER NUMBER ab15481

the different batch still did not yeild any results. same procedures were followed and no banding/signal was evident. please contact me on the next step. refund? thankyou,

ANSWER:

Thank you for your email. I am sorry that this antibody is still not working out for you and I am investigating this case with the originator of this antibody. I have instructed our accounting department to issue you a refund for your order for ab15481. If you have any questions regarding the refund, please contact accounts@abcam.com

BATCH NUMBER 96299 ORDER NUMBER 71416

DESCRIPTION OF THE PROBLEM High background, no signal, DOESNT WORK

SAMPLE human and mouse as well as recommended MCF-7

SECONDARY ANTIBODY santa cruz

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED mcf7, cb7, cb3

ANTIBODY STORAGE CONDITIONS all protocols followed

BLOCKING CONDITIONS see protocol emailed to me

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ADDITIONAL NOTES i ran western blot following my own protocol as well as one supplied by you, which was almost identical as well as a make-shift inbetween to trouble shoot.

ANSWER:

Thank you for contacting us and for taking your time to fill in the Questionnaire. We are very sorry to hear that you are having difficulty with this antibody and you can't get a signal in Western and IHC.

Certainly, we can offer you a different batch (78293) for testing. This is to inform you that the new order number for this replacement vial is 77509.

We hope that this batch will work nicely. We apologize for any inconvenience caused. If you need anything further or any help then please let us know.