Anti-heavy chain cardiac Myosin antibody [3-48] (ab15)

Dear technical team,
Our customer received ab15 today but it is turbid so wants to check this is okay.
She has beed used ab15 since 2010and said this is the first time that antibody is turbid like this.
I have attached picture and please take a look at it.
Best regards,

ANSWER:

Thank you for contacting us.

This antibody is not affinity purified, it is an IgG fraction, and the turbidity can vary from batch to batch or even from one preparation to another.

I would encourage you to use it normally, and in case you encounter any problem with it, please do not hesitate to contact us and we will be happy to help you out.

¿Qué isoformas de la proteína reconoce el Anti-heavy chain cardiac Myosin antibody [3-48]?

ANSWER:

Gracias por contactarnos.
Este anticuerpo es capaz de reconocer las isoformas alpha y beta de la proteína (VI y VII).
Si tiene cualquier otra consulta, no dude en volvernos a contactar.

Dear responsible, I want inquire if your beta-cardiac myosin heavy chain peptide (ab66470) is tested on western blotting? I want a mouse monoclonal antibody against MYH7 protein, and MYH7 control protein for my WB.   I did not find a monoclonal Ab in your company, but I found a competitor's product (mouse monoclonal antibody). Can I use your beta-cardiac myosin heavy chain peptide (ab66470) as a control protein for the Ab. If you have any other suggestions please inform me.  

ANSWER:

Thank you for contacting us.

Unfortunately, I would not recommend using ab66470 as a positive control in Western blot: It is a synthetic peptide derived from within residues 600 - 700 of Human beta-cardiac myosin heavy chain. This peptide was used as an immunogen for an antibody and is only 20 amino acids in length. Therefore, it is hardly suitable for Western blot. I apologise for the misleading information on our datasheet.

Indeed, we do have two anti-MYH7 (heavy chain cardiac Myosin) antibodies available in our catalog: ab15 and ab50967 which are guaranteed to react with myosins with the indicated affinities. All tested applications and species are specified on our datasheets, which are updated as soon as any new information is brought to our attention. Please find more detailed information here: Click here (or use the following: http://www.abcam.com/index.html?datasheet=15). Click here (or use the following: http://www.abcam.com/index.html?datasheet=50967).

Although we do not have a suitable purified control protein available, I would suggest one of the following lysates. The above mentioned antibodies have been shown to give positive signals with these tissues. Furthermore, these lysates can be of use in future experiments as a positive control for other proteins than myosin as well. ab3869: Click here (or use the following: http://www.abcam.com/index.html?datasheet=3869). ab30291: Click here (or use the following: http://www.abcam.com/index.html?datasheet=30291).

I am pleased to let you know that all our products are covered by our Abpromise: We are happy to offer technical support, and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

Furthermore, we do not currently have an image of ab15 in Western blot. Therefore, I can offer a discount off a future purchase if you buy ab15 now, use it Western blot and submit an image of your results to us in the form of an Abreview. The discount would be to the value of 1 free primary antibody. If you are interested in details of this offer, I would be pleased to send you more information on request.

I hope this information is helpful to you. Please do not hesitate to contact me if you need any further advice or information in this regard.

BATCH NUMBER 336389 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Non-specific staining

SAMPLE human cardiac derived progenitor cells

PRIMARY ANTIBODY ab 15 - anti cardiac myosin 1:400 30 min - 37C wash steps - 3x (Tris)

DETECTION METHOD immunfluoreszenz

POSITIVE AND NEGATIVE CONTROLS USED Negative controlls: primary cells: human mesenchymal stem cells, chondrozytes, cell lins: 293 T all positive!

ANTIBODY STORAGE CONDITIONS Aliquots at -20C

FIXATION OF SAMPLE 2 % Formaldehyd

ANTIGEN RETRIEVAL ---

PERMEABILIZATION STEP Triton 0,5% (in blocking buffer)

BLOCKING CONDITIONS 10 % goat serum (with Triton) 30 min 37C

SECONDARY ANTIBODY goat anti mouse Cy3, [another company] 1:500 30 min - 37C

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? we used different negative controlls

ANSWER:

Thank you for your enquiry. I am sorry to hear you are having a problem with ab15.

Your protocol is very good and the only two steps which I would like to suggest to alter are the following:

-try to fix the cells with 4% PFA for 5 and 10 minutes, as it may be that currently the fixation is not suitable for the protein and the antibody binds to all the tissue

-Wash the cells with PBS 4 times in between each step, except after blocking.

-try diluting the primary antibody more and incubating longer, so that a targeted binding is achieved; I would recommend trying 1:800, 1:1000, 1:2000, 1:5000 at 4C

-dilute the secondary more, trying up to 1:2000.

Please let me know if this helps and do not hesitate to contact us for further advice if you still experience problems; I would be happy to offer you a replacement vial or refund if you purchased the antibody in the last 120 days.

I have been using the ab15 antibody against the full length native myosin heavy chain. As other people who have written enquiries, I have also seen the multiple bands. I was wondering if you had mapped the epitope to a specific region of the protein being it is 223 kD. This would be extremely useful to me...even if you could tell me whether it recognized the N or C terminus.

ANSWER:

Thank you for your enquiry. I apologise for the long delay in responding to your message. It has taken some time to obtain more information from the laboratory.

Unfortunately, the epitope recognised by this antibody has not been mapped. However, we do know that the antibody is alpha and beta heavy chain specific. During its development, on Western blots and in ELISA, the antibody was found to react with purified chains of atrial and ventricular myosins isolated from human cardiac tissues as well as those of several other mammals. It binds to an A-band of thick filament of cardiac and skeletal muscle myosins. It also cross reacts with heavy chains of human myosin isolated from slow skeletal muscle, fast skeletal muscle, and eye muscle. It does not react with myosin light chains, human platelet or smooth muscle myosin heavy chains, nor does it react with any other human tissue antigens or blood cells.

I hope this information is helpful. Please do not hesitate to contact us again should you require more information.