Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [2]
- Immunohistochemistry [1]
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Validation data
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- Product number
- PA5-77321 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- TRPV6 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- For reconstitution, we recommend adding 100 µL distilled water to a final antibody concentration of about 1 mg/mL. To use this carrier-free antibody for conjugation experiments, we strongly recommend performing another round of desalting. (Zeba Spin Desalting Columns, 7KMWCO, 0.5 mL, Product # 89882)
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 50 µL
- Concentration
- 0.8 mg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of PC-3 cell lysate with TRPV6 polyclonal antibody (Product # PA5-77321) using a dilution of 1:200.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of TRPV6 (Transient receptor potential cation channel subfamily V member 6) was achieved by transfecting T-47D with TRPV6 specific siRNAs (Silencer® select Product # s30899, s30900). Western Blot analysis (Fig. a) was performed using whole cell extracts from TRPV6 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with TRPV6 Polyclonal Antibody (Product # PA5-77321, 1.5 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10000 dilution). Densitometric analysis of this western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TRPV6.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Anti-TRPV6 Polyclonal Antibody (Product # PA5-77321) and a 48 kDa band corresponding to unglycosylated monomeric form of TRPV6 was detected in T-47D (expresses high endogenous levels of TRPV6) but not in other breast cancer cell lines and was upregulated upon thapsigargin treatment (accumulates the protein in the plasma membrane upon calcium disregulation). Uncharacterised band at ~22 kDa was observed. Whole cell extracts (40 µg lysate) of T-47D (Lane 1), MCF 10A (Lane 2), MCF7 (Lane 3), membrane enriched extracts (40 µg lysate) of T-47D (Lane 4), thapsigargin treated T-47D (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1.5 µg/mL dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Antibodies designed to C-terminus of TRPV6 detects unglycosylated monomeric forms of TRPV6 [doi:10.1074/jbc.M113.469726].
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunocytochemistry analysis of TRPV6 in paraformaldehyde-fixed and permeabilized PC-3 cells. Samples were probed with TRPV6 polyclonal antibody (Product # PA5-77321) at a dilution of 1:100, and incubated with goat-anti-rabbit-AlexaFluor-555 and Hoechst.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of TRPV6 was performed using 70% confluent log phase T-47D, MDA-MB-231 and MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with TRPV6 Polyclonal Antibody (Product # PA5-77321) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2500), for 45 minutes at room temperature (Panel a,e,i: Green). Nuclei (Panel b,f,j:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c,g,k: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d and h represents the merged image showing membrane localization of TRPV6 in T-47D and MDA-MB-231, respectively and panel l represents merged image in MCF7 showing lower levels of expression for the protein in comparison to MDA-MB-231, as reported in studies. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of TRPV6 in rat dorsal root ganglion. A) Samples were probed with TRPV6 polyclonal antibody (Product # PA5-77321) using a dilution of 1:100, and incubated with parvalbumin. B) Parvalbumin stained image. C) Merged image of Panels A and B.