Anti-ADAM8 antibody (ab2475)

You are right - there is a band at 90 kDa in the positive control, but it is not very distinct, isn't it. I have already ran a no primary control with my samples and I didn't get a signal.

I attached all the blots with (I hope) enough information.

You see that I used 3 different antibodies against adam8 and I don't get specific signal. Even in the literatur I can't find much about an antibody against adam 8. So, I assume that there doesn't exit specific antibodies and that is why I'm going crazy if I buy them and they don't work. In my opinion companies should offer things which works.

Maybe you have some ideas to improve my results.

ANSWER:

Thanks for your email and for your patience. Your positive control looks very similar to the image on the datasheet. I recall that previously you were not able to detect any bands at all with ab2475 and the human brain lysate, thus why I sent you the replacements. So this is certainly an improvement.

I'm afraid that I can't really comment on what may be going on with the other Adam8 antibodies that you are using. For those, what did the originators recommend to use as positive controls? It appears that the results are somewhat similar to what you got with ab2475, but I'm not really sure based on the information that you have sent me.

The only comments that I can make at this point is to try to decrease the non-specific bands that you are seeing in your experimental samples. Try lowering the concentration of both the primary and secondary antibodies even more. There is the possibility that Adam8 is not expressed in your samples. From information that I found, Adam8 is expressed in osteoclasts and in neurons, astrocytes, and microglia during CNS neurodegeneration. Here are some general references that should be helpful for you:

Choi SJ et al. ADAM8: a novel osteoclast stimulating factor. J Bone Miner Res 16:814-22 (2001). PubMed: 11341326

Schlomann U et al. Tumor necrosis factor alpha induces a metalloprotease-disintegrin, ADAM8 (CD 156): implications for neuron-glia interactions during neurodegeneration. J Neurosci 20:7964-71 (2000). PubMed: 11050116

Yoshiyama K et al. CD156 (human ADAM8): expression, primary amino acid sequence, and gene location. Genomics 41:56-62 (1997). PubMed: 9126482

Yoshida S et al. Molecular cloning of cDNA encoding MS2 antigen, a novel cell surface antigen strongly expressed in murine monocytic lineage. Int Immunol 2:585-91 (1990). PubMed: 1982220

Please do let me know if you have any additional questions.

I already had contact with your technical support because I ordered an antibody which doesn't give a specific signal. Then I ordered the positive control to find the specific signal. Then I got a replacement because something must have happened to the antibody. So, now I carried out some western blots (see attachment) and I didn't get specific signals. I attached a protocol with the important information and pictures of the blots.

I have to say that it is an impudence to sell things which don?t work!!!!!! I bought an antibody for 350? without getting the appropriate signal and then I bought a positive control for 350?! Your support is good and you make an effort for your customers but anyway you can?t sell such things!!!! It is not okay if I get my money back and you still offer this antibody.

If you have some ideas to optimize the western blot, I will follow it.

So, I hope we find a solution.

ANSWER:

Thank you very much for your email and for your patience. We value your feedback and take your comments very seriously. A couple of things that I noticed from your results - I do see a band at 90 kDa in your positive control, but there is no band at 90 kDa in your experimental samples. In the positive control lanes there are a couple of non-specific bands but the band at 90 kDa appears to be the strongest. Have you tried decreasing the concentration of the secondary antibody? Have you run a no primary control (secondary only) with the positive control and your experimental samples? I'm curious to see if you get any non-specific binding. I recall from before that you did not see any signal at all with ab2475 and the human brain lysate.

I do remember that you were also working with an Adam8 antibody from R&D, have you tried that antibody again? What were the results in comparison to ab2475?

Thank you and I look forward to hearing from you.

no problem. I know that mails often get lost. I think that I would prefer a refund. Exept you say that you have a more specific antibody. What do you say to my blots??? Were you able to open and read the word file? What is your comment to the positive control? Isn''t it unusual that I didn''t even get a signal with anti-b actin???

I am away on leave from 13. to 21. may.

ANSWER:

Thank you for your message. After reviewing your results with the different Adam8 antibodies that you have tried, we're not sure that the R&D antibody results are reliable as the 88kDa band is very weak compared to the other bands. You also mentioned that you couldn't get another Adam8 antibody (to a different domain) to work either.

There are a couple of different options. I can send a replacement vial of the human brain lysate to try, or I can give you a refund. Please let me know what you would like.

I followed the instructions I got from you!!! I even ordered the "positive control", the human brain lysate (ab7918). I loaded 20 µg as recommended. used the primary antibody 1:1000. I ran a secondary control (no primary antibody) --> no bands appeared! I'm really not sure if the antibody works, because I only see unspecific bands!!!!! see attachment for the image what do you say????

1. On what material are you testing the antibody in WB? • human • isolated placental cells (primary trophoblasts, endothelial placental cells, cell lines (choriocarcinomacells)) I scrabbed the cells with a mixture of 1:1 Lysisbuffer (0,01 M Tris pH 7,4, 1 % SDS, 1mM Na-Orthovanadate, Proteaseinhibitor) and Laemmlibuffer (Elektrophoresis Sample Buffer, SIGMA) • tissue extract (TP, FTP) à tissue was hackled in Lysisbuffer with ultra turax, centrifuged and the supernatant was diluted with Laemmlibuffer • I heated the probes 5 min, 95°C and loaded 70 mg per cell type

2. Primary Antibody (ab2475) First I followed the protocoll and diluted the antibody 1:500. The result was a blot that looks like a gel which is stained with Coomassie!!!! According to your recommendation I diluted it 1:1000 and the results are attached. • goat • 1:1000 (1mg/ml) • 1h, room temperature • wash 3x10 min

3. Secondary Antibody • Anti-goat IgG-HRP antibody (R&B System, HAF109) • produced in donkeys • 1:1000, 1h at room temp, wash 3x10 min • I don’t think because I used this one for the first antibody (R&D system, AF1031, “Anti-human ADAM8 Ectodomain Antibody”), as well.

4. What detection method are you using? Super Signal West Pico Chemiluminiscent Substrate, PIERCE, #34080

5. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? I ran a no primary control and I didn't get any bands --> so the secondary antibody is okay •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) à I did exactly the same – incubation over night, 4°C •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) à yes, after both ab 3x10 min with 0.1% Tween-20 in TBS •At what size are the bands migrating? Could they be degradation products of your target? à that’s exactly the problem. Adam8 gets cleaved and the literature describes 120 kDa, 90 kDa and 60 kDa products but I don’t know which is the predominant form in my cells

ANSWER:

Thank you very much for the information that you have provided. I am concerned that you got absolutely no bands in the positive control. Your protocol seems fine and you should have expected to see a band at aprroximately 90 kDa with the brain lysate.

I can offer you a different vial of ab2475 to try (as perhaps your vial went off during shipping) or I can offer you a refund. Please let me know and give me either the Abcam order number or the purchase order number used for the order.

You mentioned the R&D antibody, AF1031, “Anti-human ADAM8 Ectodomain Antibody” - what size bands did you get using that antibody?

Thank you, and I look forward to hearing from you.

Thanks for your quick reply. Here you get all the information

1. The problem is that I get lots of bands and I can’t find the specific band. I already used another antibody (R&D system, AF1031, “Anti-human ADAM8 Ectodomain Antibody”), so I have an idea in which cells I can detect Adam8 and in which cells not. Anyway, with the first antibody I couldn’t identify the specific band. So I tried a new one (abcam, ab2475).

2. On what material are you testing the antibody in WB? • human • isolated placental cells (primary trophoblasts, endothelial placental cells, cell lines (choriocarcinomacells)) I scrabbed the cells with a mixture of 1:1 Lysisbuffer (0,01 M Tris pH 7,4, 1 % SDS, 1mM Na-Orthovanadate, Proteaseinhibitor) and Laemmlibuffer (Elektrophoresis Sample Buffer, SIGMA) • tissue extract (TP, FTP) --> tissue was hackled in Lysisbuffer with ultra turax, centrifuged and the supernatant was diluted with Laemmlibuffer • I heated the probes 5 min, 95°C and loaded 70 mg per cell type

050304 --> choriocarcinoma cell lines, film exposed 30 min 050304a --> same blot but film exposed >1h 050304b --> TP4, TP1, FTP2, FTP1 are samples from placental tissue ECA, ECV are endothelial placental cells FT, TT are primary trophoblasts cells

4. Primary Antibody (ab2475) • goat • 1:500 (1mg/ml) • 1h, room temperature • wash 3x10 min

5. Secondary Antibody • Anti-goat IgG-HRP antibody (R&B System, HAF109) • produced in donkeys • 1:1000, 1h at room temp, wash 3x10 min • I don’t think because I used this one for the first antibody (R&D system, AF1031, “Anti-human ADAM8 Ectodomain Antibody”), as well.

6. What detection method are you using? Super Signal West Pico Chemiluminiscent Substrate, PIERCE, #34080

7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) à I didn’t do yet but the band pattern of the first (R&D system, AF1031, “Anti-human ADAM8 Ectodomain Antibody”) and the second (ab2475) antibody is a different and I used for both the same antibody. With “No primary” control, you mean no incubation with prim ab but only with secondary, right? •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) à I did exactly the same – incubation over night, 4°C •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) à yes, after both ab 3x10 min with 0.1% Tween-20 in TBS •At what size are the bands migrating? Could they be degradation products of your target? à that’s exactly the problem. Adam8 gets cleaved and the literature describes 120 kDa, 90 kDa and 60 kDa products but I don’t know which is the predominant form in my cells

8. Optimization attempts • Only one time because I don’t have so much antibody

9. I don’t have positive controls. I carried out microarray analysis, RT-PCR and I know the distribution of the RNA in my different cell types. Now I want to confirm this results with western blot. Unfortunatly, this doesn’t work.

I know that I have to optimize western blott results but with this antibody I get only unspecific bands. The blot looks like a gel which is stained with Coomassie. And I have no idea where I can find the specific band and so I don’t know what I should change.

ANSWER:

Thank you for sending me the details of your protocol. To try to get rid of the non-specific bands that you are seeing, I would like to suggest the following. Decrease the amount of primary that you are using (try 1:1000) and decrease the concentration of the secondary antibody. Also, please run a secondary control (no primary antibody). I also strongly suggest running a positive control. We recommend human brain lysate (ab7918); a picture of the blot using human brain lysate and ab2475 is located on the datasheet and a band at approximately 90 kDa is seen. It's very important to run a positive control in order to determine if the antibody is working correctly.