Anti-BMP3 antibody (ab71500)

Dear Tanya,

Nice to hear from you, here are the customer's answers:

SAMPLE: Rat livers and growth plates. Q1.1: Is the customer using primary culture of rat liver? Could you please clarify what the experimental conditions are i.e. growth plates?

I used rat liver lysate ant not liver culture.

The experimental conditions:

Lysis buffer: 200 µl extraction lysis buffer (sigma)

1 µl protease inhibitor cocktail

0.12 µl DNAse

Add 200 µl of lysis buffer to every 20mg tissue

Homogenize tissue for 1 minute.

Incubate 15 minutes on ice.

Centrifuge for 1 minute at 11,000 rpm.

Take effluent for protein assessment.

Take wanted amount of protein (100 µg) and add sample buffer (x1) 1:2 ratio.

The experimental conditions are the same for growth plates and livers.

Q1.2: Normally, liver has very high levels of proteins so the degradation of the target protein (BMP3) due to proteinases is a high risk. Therefore it is essential to use a proteinase cocktail? Can you confirm what inhibitors the customer used in the lysis buffer?

I used commercial protease inhibitor cocktail (Roche). The protease inhibitors included in the cocktail are: Aprotonin, Leupeptin, Pepstatin and PMSF.

Also, we see a very strong protein staining in the liver lane (in ponceau staining).

Q1.3: Due to the high intracellular protein concentration in hepatic cells, it is also important to block with 5 or 10% BSA.

Block is used to prevent unspecific binding of the antibody to empty parts of the membrane.

Our problem was that we had no staining at all. Also, one can decide to use BSA block or milk block. We decided to use a milk block.

2. POSITIVE CONTROL: Positive control- Rat liver- showed no bands. Q2.1: This antibody has been tested on 3T3 cell lysate as the datasheet and the WB image indicates, therefore this type of lysate is the positive control for this antibody. Has the customer used 3T3 cell lysate? If not, then I would suggest running it alongside with the samples.

We did not use 3T3 cell lysate as a positive control. However, the antibody did not give any staining in my tissue, so I don't see why it is relevant to test it in your cell line. Also, it is well known that antibody stain is much harder to achieve in primary tissue lysate than in cell line lysate.

For the next western blot I am willing to try and find a 3T3 cell lysate as a positive control to test this antibody.

ANSWER:

Thank you for your reply on behalf of your customer. Tanya is away this week and I would like to reply on her behalf to prevent any delays in replying to your customer. I really appreciate the customer providing some details of their experiment, as this helps us understand the source of the problem; if a product is of poor quality will not hesitate to remove it from the catalogue, therefore detailed data is essential for us to determine this.

I hope I have understood this case correctly and to be able to provide further input into this matter.

It is really good to hear the customer uses protease inhibitors in the lysis buffer as this is essential, and that he tried blocking with milk. The blocking buffer can affect the binding affinity of the antibody, in some cases milk or BSA bind the antibody and therefore prevent total binding to the membrane so it can be worth trying BSA.

I think it is very likely that the customer has no signal due to low levels of BMP3 in the liver lysates. A literature search on this protein in this tissue did not return any references to western blotting data of BMP3 in liver, though mRNA levels have been reported to be elevated in hepatitis C virus (HCV)-associated cirrhosis. I think it is essential to have the positive control of 3T3 lysate to establish that the procedure is working well.

I hope these recommendations will help your customer; if the antibody does not work in 3T3 please let us know and we will apply the Abpromise to the customer.