Anti-Androgen Receptor antibody [AN1-15] (ab2742)

 Thanks for your kindly reply, after I contacting with this customer, and passed your information to her, she modified her experiment step as your suggestions, however the results still show wrong band size, I attached his data in this letter, she also replied you questions as follow: ·         Species: rat ·         What’s cell line or tissue：tissue ·         Cell extract or Nuclear extract: Cell extract ·         Purified protein or Recombinant protein:  Purified protein   Could you please offer another suggestions to her? Thanks for your kindly help.   Best regards    

ANSWER:

Thank you for your email. Unfortunately I have not received the attachment. Could you please send it again? Can you please confirm also what kind of tissue has the customer tested in the end - also some heart or skeletal muscle? Did the BSA blocking change anything in the pattern of the observed bands? The only other possibility I do currently see, is that the buffer is maybe not compatible with the molecular weight marker. Indeed, the proteins in the molecular weight marker can migrate very differently depending on the buffer used. Please see as an illustration for this the image on the datasheet of ab116028. http://www.abcam.com/index.html?datasheet=116028 I can recommend therefore to check on the specification sheet of the molecular weight marker if there is any information available in this regards. I am looking forward to receive the attachment and the comments of the customer to the questions above. Thank you for your cooperation! I wish you a great day!

          Dear scientific support team:   Our customer raised an inquire about Anti-Androgen Receptor antibody [AN1-15] (ab2742). She got a result with wrong band size. The predicted size is 110 kDa and she obtained 170-kDa bands. Her antibody was expired in 6 months of purchase. Therefore, could you please help her to check her protocol and give her suggestion?   I attach her protocol with this mail.   Thank you for your assistance.   Best regards,      

ANSWER:

Thank you very much for having submitted the customers questionnaire. I am very sorry that the customer has experienced problems with this antibody. I have looked through the protocol provided, and would like to make the following suggestions: 1.) I am not sure what tissue the customer exactly has used. Can you please confirm what tissue it was and whether he is sure that the protein is expressed? Else I can recommend to run a positive control with either heart or skeletal muscle tissue. 2.) In order to exclude that rat immunoglobulins are seen in the Western blot at around 150kD, I do strongly recommend a "no primary" control to the customer. Indeed, the amount of bME might have not been sufficient to reduce all the rat immunoglobulins and this could explain a band at 150kD. 3.) If indeed the band at 150kD is background, the specific band is still not visible. I would therefore recommend to the customer to try also BSA blocking in parallel. As you know, changing the blocking buffer can significantly impact on the results as seen also on the Western blot image on the datasheet of ab9385. I hope this suggestions help to find the origin of the problem. Please do let me know how the customer is proceeding. I am looking forward to hear back from you.

Dear Tech support

One of our customers has ordered ab9474. The customer wishes to know what are the chances of the antibody working With Mouse samples? Also, is there a positive control available for this antibody? Thanks in advance for your kind help.

Regards,

ANSWER:

Thank you for your interest.

It is very likely that ab9474 works in mouse but we have no data yet. According to Blast search, the homology between human and mouse AR is very high.

http://www.uniprot.org/blast/uniprot/2011101050RXMELAA8

Furthermore, we have some alternative antibodies against AR in our catalogue which proved to recognize mouse AR: ab2742, ab74272, ab3509, ab52615

Normal brain, epididymis, prostate, seminal vesicle; or skin and prostate cancer can be used as good positive control.

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

What is the isotype of this antibody? I would like to use it in IP. If you do not have the specific isotypeDid you use Protein A or Protein G beads?

ANSWER:

Thank you for your enquiry.

It appears the subclass of this antibody is unknown and the product is total IgG. I would recommend using Protein G only because it is the better bead to use for most subclasses of Rat IgGs.

In addition, below you will find a reference that used this product in IP assays as well as protocols we have specifically for this product.

Please let me know if I can provide further assistance.

Biochem. Biophys. Res. Comm., 177: 488-496, 1991. (IP protocol well outlined) PubMed link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2043134&dopt=Abstract

Protocol for use of Monoclonal Anti-Androgen Receptor

Western Blot

1.After blotting, block filters for 60 minutes with 5% non-fat dried milk, 0.2% Tween 20 in TBS pH 7.5 (DILUENT).

2.React the blot with anti-Androgen Receptor antibody diluted in DILUENT overnight.

3.Wash 3 x for 5 minutes, in TBS/0.2% Tween-20 ("Standard Wash").

4.React with anti-rat biotin diluted in DILUENT for 1 hour.

5.Standard Wash.

6.React with Avidin:HRP for 1 hour.

6.React with chemical substrate to effect. For maximum

* The procedure listed above is intended only as a guide. The results that you obtain may vary depending on experimental conditions and technique. No warranty or guarantee of performance of the above procedure is made or implied. Use good laboratory practices, handle all materials carefully.

The products and procedures are for in vitro experimental use only and are not intended for use in humans or clinical diagnosis.

General Immunoprecipitation Protocol

1. Sample Preclearing a. Wash protein A- or G-Sepharose or protein L-Agarose with 10X volume of RIPA buffer. b. Vortex and centrifuge for 1’ in a microfuge. c. Resusupend the pellet in the original volume that the protein A or protein G matrix was in. d. Add protein A- or G-Sepharose or protein L-Agarose to the sample and incubate at 4°C for 30 minutes with shaking. e. Centrifuge for 1’ in a microfuge .to pellet adsorbed nonspecific proteins and insoluble material. Discard.

2. Incubation with primary antibody a. Add primary antibody to precleared sample and incubate for 1 hour at 4°C with gentle agitation. b. Add protein-A or –G Sepharose beads and incubate 1 hour at 4°C with gentle agitation. c. Centrifuge for 1’ in a microfuge. Wash the pellet in 1 ml of RIPA buffer. d. Repeat two times.

3. Removal of antibody-antigen complex a. Resuspend immunoprecipitate in a buffered solution with either 1% SDS and 15 mM ß-mercaptoethanol or 8M urea. b. Heat at 90-100°C for 5-20 minutes with occasional vortexing. c. Centrifuge for 1-2 minutes in a microfuge. d. The supernatant is ready to be analyzed by Western blot.

The procedure listed above is intended only as a guide. No warranty or guarantee of performance of above procedure is made or implied. Always use good laboratory practices and handle all materials with care.

what is the epitope sequense within the AR used for this antibody?

ANSWER:

Thank you for your enquiry. The immunogen used was a 241 amino acid (331-572) fusion protein derived from human androgen receptor. If you have any more questions, please contact us again.