Anti-Ubiquitin antibody (ab14372)

Thank you, but my question was when was each antibody first available for sale from Abcam?  I think I was told one was first available in 2004, and the other became available in the late 1990s. Thank you,  

ANSWER:

Thank you for your quick response and I am very sorry for misunderstanding your original question.

I can confirm that we published these products in different years:

- ab411: launched on 24-Aug-99,

- ab14372: launched on 05-Oct-04

If you need anything further or any help then please let m

Thank you very much for the information sent in this and the accompanying email.  However, I did not see in the information an answer to my second question.  Would it be possible to provide information on the date of first availability of each of the two antibodies I purchased  as well, for my records? I was told that information was also available and would be sent.  Thank you very much. Sincerely,    

ANSWER:

Thank you for your response.

Regarding the availability of these two batches:

- ab411 (212534) - sold out but we have new batches in stock (1130964)

- ab14372 (199361) - sold out but we have the same bleed and purification in stock (GR23699-4 and GR23699-5)

I hope this helps and if I can assist further, please do not hesitate to con

and then, do you think that i can't use it as positive protein control in human cells in WB? thanks

ANSWER:

Thank you for your enquiry.

I can confirm that this product can be used as a detection antibody in WB. However, the product itself may not be suitable for use as positive control as the molecular structure may be too small and will run out of the gel. I would suggest using yeast cell lysates as an alternative positive control.

I hope this information will be helpful.

Have a nice day.

Thanks for your response. But I must say that I am not in the position to test the quality of your products. To simply address your questions, your anti-Ub antibody could only pick up the signal of free ubiquitin but not ubiquitin-conjugated proteins in western blot with 1:1000 dilution, following the standard procedure of western. The materials used are protein products of in vitro ubiquitylation. After re-probing the same blot with an anti-Ub antibody from Chemicon, we detected very good signals of ubiquitin conjugates. I hope this information can ensure you that your anti-Ub Ab is the one we need in our research.

ANSWER:

Since the Ubiquitin is a small 76-residue protein (8.5kDa), we prepared this antibody by immunizing rabbits against ubiquitin conjugated Rabbit IgG. It was tested by immunoblot against total cell extract from yeast. Dilution of the antibody between 1:200 and 1:1,000 showed strong reactivity with Ubiquitinated proteins. The dilution of 1:1000 you used is actually on the low end of concentration we suggest.

I assume there is only titer difference between its reactivity to free ubiquitin and that to conjugated ubiquitin. You could run an Elisa by coating free ubiquitin and ubiquitin conjugated proteins side-by-side to check the titer difference.

There is another possibility. The active ubiquitin epitopes which are recognizable by our antibody could be hidden inside of the protein which it is conjugated to. That also explains why it recognizes the free form (or shows higher reactivity), but not the conjugated form. If it is true, I have to say the antibody is not suitable for this specific ubiquitinated protein.

I hope this information helps, please do not hesitate to contact us if you need further advice.

We ordered the anti-ubiquitin antibody (ab14372) from your company in believing that this antibody can detect both free ubiquitin and ubiquitinated proteins based on the information provided by your company. Unfortunately, our western analysis showed that ab14372 could not pick up the signals of ubiquitin-conjugated proteins, whereas an antibody from another company easily detected the signals from the same blot. Obviously, I was disappointed about the quality of ab14372 and concerned about the accuracy of the information provided by your company. By the way, the data sheet says ab14372 is provided in the form of liquid. But, what we received is a lyophilized form. Because of the quality of ab14372, I request a refund on this order (#58028).

ANSWER:

Thank you for your email and I'm sorry to hear that you are experiencing difficulty with ab14372. If you would kindly provide me with some more details concerning your protocol, it would help me to determine why the antibody is not working for you. Thank you, and I look forward to hearing from you.

1. Please describe the problem (high background, wrong band size, more bands, no band etc).

2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein?

3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples?

4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step?

5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary?

6. What detection method are you using?

7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •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) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered?

9. Did you apply positive and negative controls along with the samples? Please specify.