Anti-BMP2 antibody (ab14933)

please find here below our customer's reply:

- "Reconstituted antibody has been store at -20°C", coul d you explain what this means? Did you get the product lyophilized or you just mean diluted the antibody and stored?

I’m sorry I made a mistake. Primary antibody was only aliquoted and stored at –20°C and not reconstituted.

- Could you send us an image?

You have said you have tried antibody from different company and it worked. Was it with the same protocol and lysates?

Yes, I used the same protocol but sample was different or rather I extracted protein from human dermal fibroblasts instead of mice dermal fibroblasts.

I hope this information help you.

Kind regards

ANSWER:

Thank you for providing details quickly.

The protocol seems absolutely fine. The antibody should have worked, however we are not sure why this antibody is binding non specifically. In order to know the cause of multiple bands, is it possible for you to run test with human lysates? This could be a good positive control to check if the problem is due to antibody or preparation of lystaes.

Let me know what you think?

Product code: 14933
Lot number: GR41047-2
Inquiry: OUR PO WAS: 543 1)Antibody storage conditions:Reconstitued antiboby has been store at –20°C. 2)Description of the problem: More bands 3)Sample: Protein have been extrated from mice dermal fibroblast and it has been done a total lysis 4)Sample preparation:Cells were washed several times with phosphate-buffered saline and homogenized in RIPA buffer in presence of protease inhibitors. Cellular lysates were centrifuged at 15,000 rpm for 20 min to clear cell debris, and supernatants were collected and stored at –80°C until analysis. 5)Amount of protein loaded: 30 µg proteins 6)Electrophoresis/Gel conditions: 10% polyacrylamide gel, under reducing conditions 7)Transfer and blocking conditions:Transferred to nitrocellulose. Membrane was blocked in TBS+0.1% Tween 20 + 5% non fat dry milk for 1 hr at room temperature 8)Primary Antibody:Antibody was diluted in TBS-0.1% Tween 20 + 2.5% non-fat dry milk at 1:1000 and incubation time was overnight at 4°C. 3 wash steeps and incubation of nitrocellulose with 9)Secondary Antibody:Secondary antibody (no Abcam antibody) was diluted in TBS-Tween 20 + 2.5% non-fat dry milk at 1:5000 for 1 hr. 10)Detection method: Western blots were visualized using Super Signal West Pico 11)Positive and negative controls used (please specify):I used fibroblast isolated from human skin and in this case I used an other antiboby (bought by an another company) that recognized only a band. 12)How many times have you tried the Western?: 3 times 13)Have you run a "No Primary" control?:No 14)Do you obtain the same results every time?: Yes and the number of bandes were always the same. 15)What steps have you altered?: I modified concentration of antibody both primary that secondary and the incubation time (instead overnight I tried only 1hr)

ANSWER:

Thank you for contacting us. I am sorry to hear that you are experiencing problems with this antibody.

I have checked the protocol and would like to ask few further questions for better understanding of the problem;

- "Reconstituted antibody has been store at –20°C", could you explain what this means? Did you get the product lyophilized or you just mean diluted the antibody and stored?
- Could you send us an image?
- You have said you have tried antibody from different company and it worked. Was it with the same protocol and lysates?

I look forward to hearing from you soon.

This antibody is not detecting the human purified recombinant protein by WB.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement of ab82511.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

We have a customer who is interested in AB14933. However, they were curious about the positive control. They would like to know if they can make it themselves or if they have to purchase it. If they can make the lysate buffer, do you have a suggested protocol for doing it? If they have to purchase it, do you have any suggestions as to where they can buy it from? Thank you in advance for your help,

ANSWER:

I was able to add a positive control to our catalogue, it is recombinant protein BMP2 and was tested for ab14933: the reference is ab37324

http://www.abcam.com/index.html?datasheet=37324

I hope this will help your customer,

I am not sure if Bmp2 antibody (raised in rabbit) works or not. Please refer to attached autorad scan (open in Photoshop) which includes Western for Bmp2 (top) and Bmp4 (bottom). Bmp4 antibody is also from your company. Gels were loaded from left to right in this order: Lane 1: protein marker Lane 2: 500 ng (way too much) of Bmp2 protein (top gel) or 500 ng of Bmp4 protein (bottom gel) Lanes 3-8: cell extracts Lane 9: Virgin mammary gland extract Lane 10: pregnant mammary gland extract

Bmp2 peptide is supposed to be either 18 or 36 kd; this does not seem to be the size of the Bmp2 positive control (signal at Bmp2 positive control may be artifact). At any rate: you can see there is a big difference in overall signals generated by Bmp2 and Bmp4 antibodies.

I am thinking of trying the Bmp2 antibody at a 1:250 instead of a 1:500 dilution (problem is not the secondary antibody as others in our lab have not had problems with our HRP-conjugated anti-rabbit antibody. Thank you for your interest.

ANSWER:

Thank you for your email and I'm sorry to hear that you are still experiencing difficulty with ab14933. This antibody was characterized for use in Western blotting with neonatal brain lysate and a band at approximately 45 kDa was detected. I would suggest loading less of the Bmp2 protein and incubate with the primary at a dilution of 1:500 for overnight at 4C. What are the cell extracts that you are using? And from what species? You may also want to try neonatal brain lysate as an additional positive control.

Please let me know if this helps. It would also be helpful if you could provide some additional details regarding your protocol. How were your samples prepared? What were your blocking conditions? What did you dilute the primary and secondary antibodies in? What was the concentration of secondary used and incubation period?