Anti-TGF beta 1 antibody (ab9758)

Thank you for all the help. Yes I think it might be good to try out milk also, due to time constraints and just in order to get results we skipped it because I think the ECL blocking kit contains milk. Either way, I will try milk because as you said, the result might be surprisingly good sometimes.

I would be really greatful if I could try that antibody so you are welcome to send it to:

It will be interesting to try our protocol with this antibody. Looking forward to it. Thank you for all your help!

Best regards

ANSWER:

Thank you for confirming those details. I have now arranged for the chicken polyclonal we discussed to be sent out to you at the address specified. The new order number is xxxxx(purchase order number xxxxxx). This should be with you tomorrow.

Please do let me know how you get on with your further experiments with ab9758 as well as how you find ab46780.

Until then, I wish you all the best with your writing and research.

Hello and thank you for your answer!

Any kind of help is much appriciated, I have filled your questionnaire and I'm attaching it along with 2 images of the wb results. The biggest issue is that we have multiple bands, especially near were our protein of interest should be (12 and 25 kDa). That is why we think that we can see the protein but not be completley sure. I am very busy with my thesis writing right now but will later run a longer electrophoresis in order to get better separation, and also try to use 1:1000 dilution of the prim ab to reduce the smear and perhaps also the multiple bands. Your tips in this matter would be very gladly taken into account. Tissue lysates from our WT mouse shows a band while our knockout mouse (Gai2-/-) seems to lack it (WB pic 2), which is as we have hypothezied.

Chicken polyclonal ab46780 seems interesting as it binds to the active form of our protein (as the ab we have been using), the other one you mentioned (mouse monoclonal ab64715) binds to the 45kDa inactive form which we are not interested in. I would of course first have to talk to my professor but can you do anything to the price?

Thank you for you help!

Best regards

ANSWER:

Thank you for getting back to me with that information and sorry for the delay in getting back to you.

From the blot you have shared with me, it does indeed look like you are detecting the protein as expected, especially in the human thrombocyte sample. From having reviewed the protocol used to obtain this blot there are a few suggestions I would make which may improve the non-specificity observed. As you have tried BSA and the ECL blocking agent with non-specificity observed, I would suggest also trying 5% milk blocking. This can sometimes have a surprisingly marked effect.

As you have mentioned, trying a higher dilution of the primary antibody may also help and increasing the eletrophoresis time will allow you to identify the bands of interest more clearly.

As you have had to put in quite a bit of effort in optimising this antibody I am willing to provide you with the chicken polyclonmal (ab46780) you are interested in free of charge. If you would like for me to do this could you please confirm that this is the address you would like it sent to:


I look forward to receiving your reply. Until then, I wish you all the best with your thesis writing.

Hi!

I am sorry that I haven't contacted you with a follow-up, I have been very busy with my thesis writing and experiment. We are having some difficulties but we think that we see our protein of interest. It is difficult to interpret since the antibody binds to so many things and it is not specific against only the active form of TGF-beta1. However, we will most probably continue with optimization.

Your support was very much appriciated due to the quick answers, the tips were however not helpful in this case but we of course understand that in the case of western blot it is really difficult to know what will help and what not. We have some stuff we are going to try and I am positive that we will be able to detect the protein.

ANSWER:

Thank you for getting back to me with how you have been getting on with the anti-TGF beta 1 antibody(ab9758).

I am glad to hear that you thinkyou have been detecting the expected protein. If you would like help in optimising the experiment further in order to reduce the background observed I'd be happy to help. If you would like me to do this please could you fill out the questionnaire I have attached to this email to find out the up-to-date results and protocol used. Please could you also attach an image of the results obtained.

As you have been having such problems in getting this antibody to produce good results I would be able to provide you with an alternative antibody from our catalogue if you would like to try one, such as mouse monoclonal ab64715 or chicken polyclonal ab46780. If you would likefor me to arrangethis pleasedo let me know.

I look forward to receiving your reply.

Could you please let me know whether this antibody

Anti-TGF beta 1 antibody (ab9758)

reacts with rat and opossum tissue?

Many thanks,

ANSWER:

Thank you very much for your interest in ab9758.

Unfortunately, this antibody has not been tested in opossum and the immunogen also does not show a significant homology to opossum according to NCBI Blast.

But rat shows a 100% sequence homology with the immunogen.

Therefore, I can offer a discount off a future purchase if you buy ab9758 now, test it in rat 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.

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 ab9758 in rat. I will then send a discount code. This code must be issued before purchasing ab9758 so please wait for my reply before ordering.

2. Purchase ab9758 either by phone, fax, or online (www.abcam.com).

3. Test it in rat.

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 anyprimary antibodyordered 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 ab9758 turns out to be unsuitable for rat, 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.

Dear receiver,

We bought antibodies against TGFB2 (ab36495) and TGFB3 (ab53727) to use in WB and we are a bit confused by the only predicted band size given in your datasheet; 50 kDa for TGFB2 and 47 kDa for TGFB3.

1.Are these sizes for the full size pre-pro-TGFB versions?

2.Should one not also detect the C-term mature proteins of around 12-13 kDa as well as possible dimers (approx. 25 kDa)?

3.Do these proteins maybe form tetramers of approx 47-50 kDa?

In the spec sheet for TGFB1 (ab9758) the only predicted bands are the monomer (12 kDa) and the dimer (25 kDa) so the band size info for TGFB2 and -3 was not in line with that and therefore a bit confusing.

Thanks,

ANSWER:

Thank you for your enquiry.

ab9758 - the major band at44.3 kDa corresponding to the unprocessed (pre-pro-TGF beta 1) form, the mature processed TGF beta 1 is a 25 kD homodimer composed of two 12.5 kDa subunits joined by disulfide bonds. As the Western blot image indicates a 25 kDa band is seen in the non-reducing lane and a 12 kDa band in the reducing lane. Please visit UniProt website for further information at: http://www.uniprot.org/uniprot/P01137

ab36495 - the major band at 47.8 kDa corresponding to the unprocessed form;the mature (processed)can form homodimers. UniProt: http://www.uniprot.org/uniprot/P61812

ab53727 - the major band at 47.3 kDacorresponding to the unprocessed form, the mature (processed) can form homodimers. UniProt:http://www.uniprot.org/uniprot/P10600

I hope this helps and if I can assist further, please do not hesitate to contact me.