Anti-Smad3 antibody - ChIP Grade (ab28379)

First, the lab thanks you and Abcam for the lovely brownies!

Second, I have obtained A549 cells and HeLa cells for use in controls with RAW264.7 nuclear extracts. I have not done extensive studies to figure out the antibody problem. However, the data agree with the idea that RAW264.7 cells, control or treated, do not express Smad3. I have attached these results to this email if it is of interest to you and/or Abcam.

The large image file contains the contents of the wells/treatments/and antibodies used to detect the different Smad proteins and the phosphorylated Smad proteins. The second file, Abcam Smad3 signal, shows detection of a 55kD protein in A549 cytoplasmiclysates, +/-TGFb. This signal was not detected in HeLa cytoplasmic orRAW264.7 nuclearlysates, +/-TGFb. The Smad2and3 file, shows single Smad2 and Smad3 signals in the A549 lysates, but HeLa and RAW lysates show a single band. Combining the fact that the Smad3 primary amino acid sequence is shorter than that of Smad2 and I cannot detect Smad3 in Raw cells, I believe Raw cells only express Smad2. I attached the P-Smad2and3 file to show that TGFb treatment of all cell types results in the specific phosphorylation of Smad2 and not Smad3.

Since our lab does not use A549, I am not interested in increasing the signal observed in attached LICOR images. However, I think switchingblocking buffers, I used 5% milk/TBS +/-T,as you initially suggested could work for others. Again, thank you for the brownies, we will consider Abcam a reliable source of primary antibodies in the future.

Thanks again,

ANSWER:

Thanks so much for getting back to me, and I apologize for the delay as I've been out of the office.
You're very welcome for the brownies! I know that this has been frustrating and you have spent a lot of effort and time working on this project. We are grateful for the the images and for the information about Smad3 expression. I will add this information to the datasheet in order to help other researchers.
If there is anything else that we can do for you now or in the future, please do let me know. I wish you the best of luck with your research!

THank you for your help in the information. My boss gave me a go to try these antibody so I will go ahead and use your test discount on these products.

However, I will need your help again in searching for a different clones of anti-phospho smad3. The one you suggested has cross-reactivity with Smad1/5 which makes its application in FACs problematic as I won't be able to differentiate Smad3 and Smad1/5 in FACS. In addition, it would be great if the source of antibody from species different than rabbit.

I am also looking for PRKACA, Stat3 and phopho-stat3 antibody for flow cytometry. It will be really helpful if you can make some suggestions for the clones of these antibody. And if you can find antibodies generated from different species for these genes would be great.
THank you very much

ANSWER:

DISCOUNT CODE FOR ab28379:**********
Expiration date: *************

I am very pleased to hear you would like to accept our offer and test ab28379 in flow cytometry. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for flow cytometry and 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: www.abcam.com/collaborationdiscount.

THank you for your help in the information. My boss gave me a go to try these antibody so I will go ahead and use your test discount on these products.

However, I will need your help again in searching for a different clones of anti-phospho smad3. The one you suggested has cross-reactivity with Smad1/5 which makes its application in FACs problematic as I won't be able to differentiate Smad3 and Smad1/5 in FACS. In addition, it would be great if the source of antibody from species different than rabbit.

I am also looking for PRKACA, Stat3 and phopho-stat3 antibody for flow cytometry. It will be really helpful if you can make some suggestions for the clones of these antibody. And if you can find antibodies generated from different species for these genes would be great.
THank you very much

ANSWER:

Thank you for your reply.

The only other phospho-smad3 (S423+S425) we have are:

ab11825 - http://www.abcam.com/Smad3-phospho-S423-S425-antibody-ab118825.html

ab52903 - http://www.abcam.com/Smad3-phospho-S423-S425-antibody-EP823Y-ab52903.html



Please let me know which one you would like to test and I will then send you the testing discount codes for it.

Also for the other antibodies, below are some selections:

PRKACA,

ab124390 - http://www.abcam.com/PRKACA-antibody-X1-ab124390.html

Stat3
ab119352 - http://www.abcam.com/STAT3-antibody-9D8-ab119352.html

phospho-Stat3
ab30647 - http://www.abcam.com/STAT3-phospho-S727-antibody-ab30647.html
ab30646 - http://www.abcam.com/STAT3-phospho-Y705-antibody-ab30646.html



Above are only a few of the antibodies that we have for these targets, so please feel free to search and see if you can find a more suitable alternative. Also non have been tested in flow cytometry and so would qualify for a testing discount code.

Please let me know if there is anything else I can help you with.

Looking for antibodies against TGF beta recepto1 and smad 3 (phospho +total) that will work in flow cytometry and also the appropriate isotype control.

ANSWER:

Thank you very much for contacting Abcam.

The isotype control that I would recommend for your flow cytometry experiments is:

http://www.abcam.com/Rabbit-IgG-ChIP-Grade-ab37415.html



Below is the infromation about the testing discount program, you would be eligbale to get a testing disount for all 3 antibodies:

http://www.abcam.com/TGF-beta-Receptor-I-antibody-ab125310.html
http://www.abcam.com/Smad3-phospho-S423-S425-antibody-ab51451.html
http://www.abcam.com/Smad3-antibody-ChIP-Grade-ab28379.html



To our knowledge, ab125310/ab28379/ab51451 has not been tested in Flow Cytometry. Therefore, I can offer a discount off a future purchase if you buy ab125310/ab28379/ab51451 now, test it in Flow Cytometry and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free PRIMARY ANTIBODY for each antibody that is tested in Flow Cytometry.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab125310/ab28379/ab51451 in Flow Cytometry. I will then send a discount code. This code must be issued before purchasing ab125310/ab28379/ab51451 so please wait for my reply before ordering.

2. Purchase ab125310/ab28379/ab51451 either by phone, fax, or online (www.abcam.com).

3. Test it in Flow Cytometry.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: http://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any each PRIMARY ANTIBODY ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab125310/ab28379/ab51451 turns out to be unsuitable for Flow Cytometry, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

I suppose it is possible that the cell line I am using does not express Smad3. I have shown that untreated RAW264.7 cells, a murine macrophage-like cell line, express Smad2/3 and that TGFb treatment results in nuclear accumulation of phosphorylated Smad2/3. The antibodies I used for those studies do not specifically detect Smad2 or Smad3, rather the antibodies do cross react with these Smad family members. The antibodies I used detected a single band of approximately 50-55 kDa. I think it is possible, given the Smad3 blots and the data I described,that RAW cells only express Smad2.

Could you recommend a few cells lines that only express Smad3 or Smad2 and Smad3? I could test these using the Abcam Smad3 antibody in question, and the other antibodies I have for Smad2/3. I was thinking about using HeLa cells given the HeLa stained with Smad3 (ab28379) on the website. However, there is no Smad3 western blot shown with HeLa lysates.

Thanks in advance,

ANSWER:

Thanks for your reply.
I agree that running a known positive control is a good idea in this case, just in case the cells are expressing Smad 2 exclusively. Ab28379 has been tested in Western blot against Jurkat cell lysate, A549 lysate, and MDA231 cells, though if you don't have access to any of these cell lines I'll do some literature searching and see if there are others that might work.
Please let me know what you think, and keep me updated about any new results with this antibody. I look forward to hearing from you.