Anti-Neurokinin 1 Receptor antibody (ab466)

I have tried the antibody in western blot and find the 53 kD band in blood lymphocytes. However, there are also other bands between 60 an 88 kD. In other cell types (skin) the 53 kD is not found but a clear band at 85 kD. What is known about the isoforms of NK-1 (size)? In the litterature I found also published bands at 80 kD (adipocytes). In your Abcam review the user found also this higher molecular weight bands in brain tissue. Have you ever attempted to identify these? Are they more likely isoforms of NK-1Ror unspecific reactions (e.g. with other receptors)?

ANSWER:

Thank you for your enquiry.

There are published reports stating that the substance P receptor/neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. Research has shown that different tissues express different ratios of the two isoforms of the NK-1 receptor.

However, the originator of the antibody has not done any extensive testing to determine individual expression patterns of these receptors in various tissues or to further characterize the bands recognized by the antibody in western blot.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

Is the antibody directed against an extracellular portion of the receptor? Can i use it for sorting live NK1R cells with FACS?

ANSWER:

Thank you for your enquiry.

Unfortunately I cannot comment on whether the antibody recognises the intra- or extra-cellular domain as I am not an expert in the field. I can however tell you that the immunogen for this antibody is the at the N-terminus (aa residues 1-17). Furthermore given that the target protein is at the cell membrane you can perform cell sorting on live cells. For a detailed protocol for this antibody please follow the link below.

http://abcam2/index.html?pageconfig=protocols&pid=248&intAbID=466&strTab=protocols&mode=prot

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

Dear Jennifer,

you should be able to read it now.

ANSWER:

Thank you for re-sending the information, I was able to open the attachments. Below is the FACS protocol which the researcher who tested it for Novus used. They also used lot C, so the vial that your customer received should be alright. I hope this is helpful, and if there are still problems, please let us know.

FACS Analysis 1. Seed cells into small (T-25) flask. Add 5-10 ml media. Allow cells to grow to 80-100% confluence. 2. When flasks are confluent, empty media and add new media containing appropriate drug, hormone, or cytokine dose. 3. Allow flasks to incubate for 24 hours. After incubation period, remove 2 ml of media and place 1 ml into one of two eppendorf tubes. 4. Empty off remaining media. Wash with 5 ml 1X PBS. 5. Add 2 ml PBS with azide (1% Goat serum). Scrape cells using a cell scraper. 6. Transfer cells to appropriate labeled FACSCAN tubes. Vortex all tubes 7. Incubate on ice for _ hour in the PBS with azide (1% Goat serum). Vortex all tubes after incubation. 8. Centrifuge at 1400 RPM for 5 minutes. Empty off media. 9. Resuspend in 1 ml of a 1:3,000 primary antibody solution (NB 300-119) [1 ul antibody per 3 ml PBS with azide (1% Goat serum)]. 10. Incubate on ice for 1 hour. Vortex all tubes after incubation. 11. Centrifuge at 1400 RPM for 5 minutes. Aspirate (or decant) supernatant. 12. Wash twice with 2 ml ice-cold PBS with azide (1% Goat serum). 13. Aspirate (or decant) supernatant and place tubes on ice. 14. Resuspend in 1 ml of a 1:500 secondary antibody solution [2 ul antibody per 1 ml PBS with azide (1% Goat serum)]. 15. Vortex all tubes. 16. Cover with foil. Incubate for 30 minutes on ice. 17. Vortex after incubation. Centrifuge at 1400 RPM for 5 minutes. 18. Aspirate (or decant) supernatant. Wash twice with 2 ml ice-cold PBS with azide (1% Goat serum). 19. Aspirate (or decant) supernatant. Resuspend in 2% paraformaldehyde. 20. Read on flow.

Has this antibody been used in rat tissue?

ANSWER:

This antibody has been used in rat tissue for western analysis with success(other applications are unknown)