Anti-MyoD1 antibody [5.2F] (ab16148)

testing ab34360 and ab16148 in flow cytometry
ab47052: check emission wavelength
send link to EasyLink kit page

ANSWER:

Thank you for your phone call.

I am going to contact the lab shortly regarding ab47052.

The link to our EasyLink conjugation kits can be found here: http://www.abcam.com/Easylink

For the information regarding the discount codes please see below:

DISCOUNT CODE: xxx for ab34360
DISCOUNT CODE: xxx for ab16148
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab34360 and ab16148 in flow cytometry. Each code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for flow cytometryand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: http://www.abcam.com/collaborationdiscount.

Thanks for your kindly reply, the product information as follow:
Lot: gr35904-2
This customer also indicated he found that the antibody molecular weight from other company shows in 45kDa, but our datasheet show in 35kDa, could you please help this customer to solve the problem?
Thanks for your kindly help

ANSWER:

Thank you for your patience.

Regarding the different reported molecular weights, I can let you know that the approximate 35 kDa on our datasheet refers solelyto the protein part of MyoD1 (as indicated for example in SwissProt Q02346, http://www.uniprot.org/uniprot/Q02346), and does not take any post-translational modifications into account. Modifications such as phosphorylation, acetylation or ubiquitination which are described for MyoD1 can alter the electrophoretic mobility of the protein in gel. Depending on the modification the band can thus run at a higher or lower size than the predicted MW.

If the customer would like to receive some protocol tips, I would recommend the following:

1) Ensure that the protease inhibitor mix is freshly prepared and contains inhibitors for all relevant protease types (for details seehttp://www.abcam.com/index.html?pageconfig=resource&rid=11379#A2).

2) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. Also, I would like to recommend not to mix the blocking reagents invarious steps of the protocol, but rather to use the same blocking reagent also in the antibody dilution buffers (i.e. BSA).

3) Dilute the primary and secondary antibody further to reduce the background and/or unspecific bands. Less antibody than 1 µg/ml might be sufficient to obtain optimal results.

This however might not necessarily explain or improvethe results the customer observed. Therefore, in this particular caseI can make a special exception and offer a free of charge replacement. May I recommend ab64159 for this? We have a blocking peptide available for it whichcould help to determine the specific band in case the results were similar to before.

http://www.abcam.com/index.html?datasheet=64159 (or use the following: http://www.abcam.com/index.html?datasheet=64159).

Otherwise, I would be pleased to arrange a replacement with any other primary antibody or issue a credit note in compensation if preferred.

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

Thanks for your kindly reply, this customer wants to know whether ab29431 (Heart (Human) Tissue Lysate - adult normal tissue) can be a positive control of this antibody or not? If not, could you please offer any suitable selection for this customer?

Thanks for your kindly help

Best regards

ANSWER:

Thank you for contacting us.

ab29431 can be used a s positive control.

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

Dear technical support team, Our customer raises an enquiry about ab16148 (Anti-MyoD1 antibody [5.2F]), according to the online datasheet this antibody’s positive control can be used rhabdomyosarcoma, this customer wants to know is there any suggestion can help she to choice the other positive control? Could you please help this customer to solve the problem? Thanks for your kindly assistance. Best regards

ANSWER:

Thank you for contacting us. I presume customer is looking for positive control in western blot.
We recommend using Heart, Skeletal muscle tissue lysates or human fetal spleen lysates.
For IHC-P sections of the above said tissues can be used as positive control.

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

BATCH NUMBER 218420 DESCRIPTION OF THE PROBLEM Multiple bands, and strongest band has the wrong size: 25kDa instead of 34 kDa. The primary antibody is a monoclonal antibody and I blasted it and only myod1 is recognized. I found article were the molecular weight was higher around 45 kDa en 55 kDa but not one says anything about a lower molecular weight for western blot of Myod1. I don't know why the brightest band is so low. SAMPLE lysate from denervated mouse muscle PRIMARY ANTIBODYAbcam/ mouse/ diluted in TBST (0,1% tween) with 1% milk(lane1) or 0,1% BSA( lane 2) or 1% BSA (lane3)/ 1/1000 / 60 minutes incubation / Wash first 5 times quick with TBST (0,05% Tween)and then 3 time 15 min. DETECTION METHOD ECL : exposure time 2 and 15 min POSITIVE AND NEGATIVE CONTROLS USED only positive denervated muscle (24 hours after denervation) ANTIBODY STORAGE CONDITIONS 4°CSAMPLE PREPARATION lysate buffer: 1x NP40 Low salt lysis buffer: 1% NP40, 50 mM TRis pH8.0, 150mM NaCL, 150 mM NaFl, 0.5mM Naorthovanadate, 50 mM B-glycerol phosphate, 0.1mg/ml PMSF + protease inhibitor solution (Mini EDTA-free protease inhibitor cocktail, Roche). AMOUNT OF PROTEIN LOADED 100ugELECTROPHORESIS/GEL CONDITIONS invitrogen precast 10%gel in MOPS buffer TRANSFER AND BLOCKING CONDITIONS transfer buffer: 5,8 g TRis Base + 2,9 gr glycine in 800 ml H20 + 200 ml MeOH + 3,8 ml 10% SDS transfer time: 60 min at 100 V blocking: overnight in TBST (0,1% Tween) with 5 % milk SECONDARY ANTIBODY Another Company IR/ donkey/ TBST( 0,05% tween)/1/10000/ 60 minutes/Wash first 5 times quick with TBST (0,05% Tween) and then 3 time 15 min.HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 WHAT STEPS HAVE YOU ALTERED?Nothing yet. I want to compare normal muscle with denervated muscle but first I have to know what the lowest band can be. ADDITIONAL NOTES In the attached file you find the blots for 2 and 15 min exposure. Every lane is denervated muscle sample but the diluent of the primary antibody is different : -lane 1: 1% milk in TBST - lane2: 0,1% BSA in TBS - lane3: 1% BSA in TBST

ANSWER:

Thank you for taking the time to contact me with this issue and your patience in awaiting a response. I am currently in contact with the originator of this product in order to gain some further information.

It would appear that in this case the predicted KDa has been derived from the “Swiss Prot” data base entry of the target (35KDa); however the cleavage fragment may be smaller than this.

Personally I would expect to see a certain amount of variation due to the molecular weight markers that have been employed.

Where there many bands below the predicted KDa this may be down to preotolytic breakdown. Whilst you have included inhibitors in the lysate itself rapid break down would still occur during the process of denervation, could I please enquire as to the steps that were taken in order to minimise this?