Anti-Prolactin Receptor antibody [T6] (ab2773)

ab25630: WB with rat liver
sees band at 25 kDa
transfer is OK, detects other proteins in the blot
Ab: 1/1000 1-2 h RT
block: overnight 4 degree
secondary: OK
antibody from other company to same protein gives same results
suggestions: WB conditions might not yet be optimal, try blocking for only 1-2 h RT, antibody incubation: overnight 4 degree
ab25631: rat is unsuitable
ab2773: WB with rat liver, sees band at 25 kDa
suggestions: WB conditions might not yet be optimal, try blocking for only 1-2 h RT, antibody incubation: overnight 4 degree

ANSWER:

Thank you for contacting us.
I am sorry to hear about the problems you have encountered.

The bands you are seeing at 25 kDa could be the light chain of endogenous IgG.

As we discussed over the phone, please try the following suggestions as it seems the WB conditions for these 2 antibodies (ab25630 and ab2773) are not yet optimal:

1) try blocking for only 1-2 h RT
2) try the primary antibody incubation for overnight 4 degree

As for ab25631, this has been shown to not react with rat samples and would probably not give satisfactory results.However, if you have access to a human sample, you could try the antibody on that.

Please let me know if the tips were helping to improve your results.

I hope this information is helpful to you, and I look forward to hear back.

Our customer has tried the proteolytic pretreatment with 0.1% trypsin but the result again was not very convincing (see attached pdf-file 'PrlR-Staining'). The file shows pictures taken from stainings with AB2773 (page2) and pictures taken from stainings with the non-commercial antibody T6 (page1, from S. Kloehn et al.). The T6 antibody is described in: Ouhtit A, Ronsin B, Kelly PA, Morel G. Ultrastructural expression of prolactin receptor in rat liver. Biol Cell. 1994;82(2-3):169-76 Okamura H, Zachwieja J, Raguet S, Kelly PA. Characterization and applications of monoclonal antibodies to the prolactin receptor. Endocrinology. 1989 May;124(5):2499-508. So you can see that the results obtained with AB2773 are still not very good. If you have any more suggestions on how the results can be imroved they will be highly appreciated. (Please notice that I am on holiday next week, but my collegue Christian Erlenhoefer will take my place during that time) Previous message:

I have a complaint about your Prolactin Receptor antibody #AB2773:

Our customer used it in IHC staining on formalin-fixed, paraffin embedded tissue sections of rat liver and pituitary.

Protocol:

Antigen Retrieval: - with 10 mM Citrate buffer pH 6.0 using a Microwave

Blocking: 5 min 0.5% Triton in TBS wash with TBS 20-25 min 0-35% H2O2 in TBS/Methanol (90/10) wash 3x with TBS 10 min DAKO blocking solution A 10 min DAKO blocking solution B wash 3x with TBS

Primary antibody: AB2773 (Lot 19068) 1/50 in antibody diluent (DAKO) for 1h at RT wash 3x with TBS

Secondary antibody: biotinylated secondary antibody from DAKO (LSAB-Kit, ready to use) for 30 min at RT wash 3x with TBS Streptavidin-Biotin-Complex (DAKO) for 30 min at RT wash 3x with TBS

Detection with AEC-Substrate Kit.

Counterstaining in Hämalaun, embedding in Kaisers Glycine-Gelatine.

Problem: no specific staining could be obtained. The same protocol was used successfully using a Ki67 antibody on rat tonsil.

Please hava a look at the attached pictures.

ANSWER:

Here is a reference that uses our monoclonal antibodies with IHC procedures:

Rl Sorenson and LE Stout. Prolactin Receptors and JAK2 in Islets of Langerhans: An Immunohistochemical Analysis. Endocrinology 136:9 pp 4092-4098. 1995

Also used in FACS analysis:

M. Gagnerault, et al. Expression of Prolactin Receptors in Murine Lymphoid Cells in Normal and Autoimmune Situations. J. Immunol., 150(12): 5673-5681, 1993.

The customer should follow these protocols along with the other information have passed onto him/her with our previous correspondence. If not, please let us know how you would like to proceed. This will work, otherwise, there is another issue and not the antibody.

Please let us know if this helps or if you require further assistance. Thank you,

do you know if the antibody AB2773 to Prolactin Receptor is specific to the long or short form of the receptor? Do you have an antibody available, that is specific for the long form only?

ANSWER:

Ab2773 has been shown to be specific for the short form around 42 kDa. The other Prolactin Receptor antibody that we currently have, ab2772, detects both the short and long forms. So unfortunately we don't have what your customer is looking for.

I would like to purchase a trial size (enough for 1 immunoblot) i.e. 20 ug of both the U5 (ab2772) and T6 (ab2773)- prolactin receptor antibodies to try out. Is this possible and can you give me a quote please.

ANSWER:

We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to test an antibody in an application for which we do not have any information, please let us know how you get on and in return we will forward a GBP10/ USD15/ EUR15 Amazon gift voucher.