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Capsaicin receptor (VR1/TrpV1), Mouse Monoclonal Antibody

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Catalog Number

M-1714

Product Info

Product Name Capsaicin receptor (VR1/TrpV1), Mouse Monoclonal Antibody
Product Description google
Mouse anti-Capsaicin receptor (TrpV1) Monoclonal Antibody (Unconjugated), suitable for WB, FC, IHC, ICC.
Alternative Names VR1; Transient receptor potential cation channel subfamily V member 1; TrpV1; osm-9-like TRP channel 1; OTRPC1; Vanilloid receptor 1; Capsaicin receptor; VR-1
Application(s) FC, ICC, IHC, WB
Antibody Host Mouse
Antibody Type Monoclonal
Specificity Antibody is specific for rat/mouse VR1 protein in westerns and immunofluorescent immunohistochemistry on mouse PEG fixed DRG tissues. Pre-absorption with immunogen obliterates positive staining. Cross reactivity with other non-VR1 proteins is minimal; cross reactivity with VR1 from other species not yet tested. This antibody clone is known to react with rat and mouse TrpV1. It is predicted to react with guinea pig due to sequence homology.
Species Reactivity Guinea Pig (Predicted), Mouse, Rat
Immunogen Description A synthetic peptide (C-GSLKPEDAEVFKDSMVPGEK) as a part of the C-terminal rat VR1 protein (aa: 819-838) has been used as the immunogen.
Conjugate Unconjugated
Purity Description Protein G purified mouse immunoglobulin
Regulatory Status For research use only.

Specifications

Product Description
Mouse anti-Capsaicin receptor (TrpV1) Monoclonal Antibody (Unconjugated), suitable for WB, FC, IHC, ICC.
Application(s) FC, ICC, IHC, WB
Application Details
Flow Cytometry: 2 ug/10^6 cells.

Western blotting: 0.5-2 µg/mL , SDS-PAGE on Bis-Tris gel 4-12%, 5% beta-mercaptoethanol, primary antibody O/N incubation in 5% skim milk/TBST. Secondary is anti-mouse-HRP, 1/6000 dilution, 2h at room temperature. Blot developed on Li-Cor C-DiGit Scanner.

IHC: Frozen or PEG embedded tissues tested (PEG embedding, see Klosen P et al (1993) J Histochem Cytochem. 41(3):455-63). Conditions tested: 1-10 µg/mL in PBS, 48 hours, followed by detection via directly conjugated fluorescent anti-mouse secondary.

Immunohistochemistry Protocol for Product Image
4% PFA perfused, formalin fume-fixed DRG tissues: Adult male rat was perfusion-fixed transcardially with 4% paraformaldehyde. The lumbar dorsal root ganglia were isolated and embedded into egg yolk medium, which was solidified in formalin fume. 30-µm-thick floated sections were prepared with a Vibratome. For pretreatment, the sections were thoroughly rinsed in 0.1M PBS (pH 7.6) and then incubated with a mixture of 0.5% H2O2 and 0.2% Triton X-100 for 30 min. Then, antigen retrieval was performed with 0.1M citrate buffer (pH=6.0) at 80 °C for 30 min. (Please note that without this step, we did not obtain any labeling). Then, the TRPV1 signal was detected using a 1:5000 dilution of the primary antibody in 2% normal horse serum (NHS) in PBS (16 h; RT), followed by a 1:500 dilution of the biotinylated secondary antibody (2 h in 2% NHS/PBS; RT) and then, the ABC Elite reagent (Vector; 1:1000 in Tris buffer; pH 7.6). The signal was visualized with Ni-DAB chromogen. The immunostained sections were rinsed in Tris buffer, mounted on microscope slides, air dried, counter-stained with cresyl violet, dehydrated and coverslipped with DPX mounting medium.
[Immunohistochemistry protocol generously provided by Dr. Szabolcs Takács & Dr. Erik Hrabovszky, Institute of Experimental Medicine, Budapest, Hungary].

Antibody not yet tested on paraffin-embedded sections. Other immunohistochemistry methods have not yet been tested but are expected to be reactive under correct conditions. Antigen recovery is suggested; the epitope is internal cytoplasmic and could be covered by internal proteins during fixation.

ICC: 4% formaldehyde fixed cells tested; requires permeabilization step as antigen epitope is intracellular. Suggested primary antibody concentration: 1-2 µg/mL.

Biosensis recommends optimal dilutions/concentrations should be determined by the end user.
Target Capsaicin receptor (TrpV1)
Specificity Antibody is specific for rat/mouse VR1 protein in westerns and immunofluorescent immunohistochemistry on mouse PEG fixed DRG tissues. Pre-absorption with immunogen obliterates positive staining. Cross reactivity with other non-VR1 proteins is minimal; cross reactivity with VR1 from other species not yet tested. This antibody clone is known to react with rat and mouse TrpV1. It is predicted to react with guinea pig due to sequence homology.
Target Host Species Rat
Species Reactivity Guinea Pig (Predicted), Mouse, Rat
Antibody Host Mouse
Antibody Type Monoclonal
Antibody Isotype IgG2b, kappa
Clone Name BS397
Conjugate Unconjugated
Immunogen Description A synthetic peptide (C-GSLKPEDAEVFKDSMVPGEK) as a part of the C-terminal rat VR1 protein (aa: 819-838) has been used as the immunogen.
Sequence C-GSLKPEDAEVFKDSMVPGEK; aa 819-838 rat VR1
Purity Description Protein G purified mouse immunoglobulin
Format Lyophilized from PBS, pH 7.4 with 3% trehalose.
Reconstitution Instructions Spin vial briefly before opening. Reconstitute in 100 µL sterile-filtered, ultrapure water. Centrifuge to remove any insoluble material. Final buffer contains no preservatives but will contain 3% trehalose and buffer salts.
Storage Instructions Store lyophilized antibody at 2-8ºC. After reconstitution, divide into aliquots and store at -20°C for higher stability. Antibody contains no preservatives. Storage at 2-8°C with an appropriate antibacterial agent. Use sterile methods! The highest purity Glycerol (1:1) may be added for additional stability when stored at refrigerated or freezing temperatures. Avoid repetitive freeze/thaw cycles.
Batch Number Please see item label.
Expiration Date 12 months after date of receipt (unopened vial).
Alternative Names VR1; Transient receptor potential cation channel subfamily V member 1; TrpV1; osm-9-like TRP channel 1; OTRPC1; Vanilloid receptor 1; Capsaicin receptor; VR-1
Uniprot Number O35433
Uniprot Number/Name O35433 (TRPV1_RAT)
Scientific Background The capsaicin receptor (VR1, TRPV1) is a ligand-activated non-selective calcium permeant cation channel involved in detection of noxious chemical and thermal stimuli. The receptor seems to mediate proton influx and may be involved in intracellular acidosis in nociceptive neurons. It is involved in mediation of inflammatory pain and hyperalgesia. Sensitized by a phosphatidylinositol second messenger system activated by receptor tyrosine kinases, which involves PKC isozymes and PCL. Activation by vanilloids, like capsaicin, and temperatures higher than 42 degrees Celsius, exhibits a time- and Ca2+-dependent outward rectification, followed by a long-lasting refractory state. Mild extracellular acidic pH (6.5) potentiates channel activation by noxious heat and vanilloids, whereas acidic conditions (pH less than 6) directly activate the channel. Can be activated by endogenous compounds, including 12-hydroperoxytetraenoic acid and bradykinin. Acts as ionotropic endocannabinoid receptor with central neuromodulatory effects. Triggers a form of long-term depression (TRPV1-LTD) mediated by the endocannabinoid anandamine in the hippocampus and nucleus accumbens by affecting AMPA receptors endocytosis (Ref: uniprot.org).
Shipping Temperature 25°C (ambient)
UNSPSC CODE 41116161
Regulatory Status For research use only.

Images, Protocols & SDS

A: Analysis of TRPV1 expression in rat PC12 cell line by Flow Cytometry. Fixing and permeabilization of cells: Absolute methanol (10 minutes in ice) and 0.1% Tween-20 in PBS, Blocking: 1% BSA, Primary antibody: Mouse Monoclonal antibody to TRPV1 (cat # M-1714-100, 2 µg per ~10^6 cells) for 30 minutes at RT, Secondary antibody: Goat anti-mouse PE labeled secondary antibody (1:100 dilution), 20 minutes in dark at room temperature. Negative control: Non-specific Control IgG, clone X63 (cat # M-1249-200, black dashed). Data and results were generated using Orflo MoxiflowTM instrument and protocols.
B: Western blot of TrpV1 in rat PC12 cell lysates (80 µg/lane). M-1714-100 detects TrpV1 protein at 95-100 kDa. SDS-PAGE: denatured and reduced; Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C (2 µg/mL); Secondary antibody: anti-mouse-HRP (1/6000) 2 hours at RT; Detection: Chemiluminiscence.
C: Immunohistochemical staining of TrpV1 in mouse dorsal root ganglia. Immunoreactivity was visualized with anti-mouse-Cy3 conjugate (red). Magnification: 20x. Courtesy P. Vilimas, Flinders University Adelaide.
D: Western blot (denatured and reduced) of TrpV1 in cell lysates of forskolin and NGF stimulated 50B11 hybrid mouse x rat DRG cell lines and NGF-stimulated PC12 cells (10 µg/lane). M-1714-100 detects monomeric TrpV1 protein at 95-100 kDa. Primary antibody: 1 µg/mL (4°C overnight). Detection: Chemiluminiscence. Courtesy Dr. D. Matusica, Flinders University.
A: Immunofluorescence analysis of TrpV1 expression in rat C6 glioma cells. Fixed (4% formaldehyde), permeabilized, and blocked (10% normal horse serum, 0.1% Triton X100) C6 cells were incubated with TrpV1 antibody M-1714-100 (clone BS397, 2 µg/mL, green) for 1 hour. Primary antibody binding was visualized with a secondary donkey anti-mouse-CF488A antibody (5 µg/mL, 1 hour incubation). Cell nuclei were stained with Hoechst dye (blue). Magnification: 100x.
B: Analysis of TRPV1 expression in C6 cells by Flow Cytometry. Fixing and permeabilization of cells: Absolute methanol (10 minutes in ice) and 0.1% Tween-20 in PBS, Blocking: 200 µg/mL sheep IgG, Primary antibody: Mouse Monoclonal antibody to TRPV1 (cat # M-1714-100, 2 µg per ~10⁶ cells) for 30 minutes at room temperature, Secondary antibody: Goat anti-mouse PE labeled secondary antibody (1:100 dilution), 20 minutes in dark at room temperature. Negative control: Non-specific Control IgG, clone X63 (cat # M-1249-100, black dashed). Data and results were generated using Orflo Moxiflow^TM instrument and protocols.

Western blot of TrpV1 in mouse brain homogenate (25 µg/lane). M-1714-100 detects bands at 90-100 kDa and 180-200 kDa corresponding to capsaicin receptor monomer and dimer. SDS-PAGE: denatured and reduced; Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C; Secondary antibody: anti-mouse-HRP (1/6000) 2 hours at RT; Detection: Chemiluminiscence.

Analysis by immunohistochemistry displaying the TrpV1-positive neurons (pseudounipolar ganglion cells, red arrows in enlargement) of dorsal root ganglia involved in pain sensation. Entire photo is presented in the inset image, and the elarged version is provided for detail.

4% paraformaldehyde-fixed, formalin-fumed, 30-µm-thick vibratome sections were used. The sections were treated with 0.1M citrate buffer (pH=6.0) at 80 °C for 30 min for epitope unmasking. Then, the TRPV1 signal was detected using a 1:5000 dilution of the M-1714-100, followed by the biotinylated anti-mouse secondary antibody and the ABC Elite reagent, using Ni-DAB as the chromogen. Tissues were counterstained with cresyl violet (lighter blue nuclei). The small compact nuclei surrounding ganglion cells (best seen around large unstained ganglion cells) correspond to satellite cells, the typical glial cells of ganglia.
[Immunohistochemistry generously provided by Dr. Szabolcs Takács & Dr. Erik Hrabovszky, Institute of Experimental Medicine, Budapest, Hungary].

Citations & References

Specific References Matusica D et al. (2020) Differentiation of the 50B11 dorsal root ganglion cells into NGF and GDNF responsive nociceptor subtypes. Mol Pain. 16:1744806920970368 Application: Rat, WB.

Bai J et al. (2018) [EXPRESS] Attenuation of TRPV1 by AMG-517 after Nerve Injury Promotes Peripheral Axonal Regeneration in Rats. Mol Pain. [Epub ahead of print] Application: Rat, WB.
General References Peng H.Y. (2008) TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats. Am J Physiol Renal Physiol. Nov;295(5):F1324-35.