710855

antibody from Invitrogen Antibodies

Targeting: STX6

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

710855

Provider

Invitrogen Antibodies

Product name

Syntaxin 6 Recombinant Polyclonal Antibody (8HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Pig, Bovine, Rat and Mouse.

Antibody clone number

8HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of Syntaxin 6 was achieved by transfecting SH-SY5Y with Syntaxin 6 specific siRNA (Silencer® select Product # s19959, s19958). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the Syntaxin 6 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Syntaxin 6 Recombinant Polyclonal Antibody (Product # 710855, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25µg/ml, 1:4000 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 Syntaxin 6.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Syntaxin 6 Recombinant Polyclonal Antibody (Product # 710855) and a 30kDa band corresponding to Syntaxin 6 was observed in the cell lines tested. Whole cell extracts (30 µg lysate) of SK-N-SH (Lane 1), SH-SY5Y (Lane 2), IMR-32 (Lane 3), HT-29 (Lane 4) and Caco-2 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of SH-SY5Y (Lane1), U-87MG (Lane 2), SK-N-SH (Lane 3) and MDCK (Lane 4). The blots were probed with Anti-Syntaxin 6 Recombinant Rabbit Polyclonal Antibody (Product # 710855, 0.5-1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A clear 30 kDa band corresponding to Syntaxin 6 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on fixed and permeabilized MCF-7 cells for detection of Syntaxin-6 using Anti-Syntaxin-6 Recombinant Rabbit Polyclonal Antibody (Product # 710855, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Syntaxin-6 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938,). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Syntaxin-6. Panel e) represents control cells with no primary Antibody to assess background.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Syntaxin 6 was immunoprecipitated using 2 æg of the Syntaxin 6 Recombinant Rabbit Polyclonal Antibody (Product # 710855) from SK-N-SH cell extracts (Lane 3) using the Dynabeads® Protein A Immunoprecipitation Kit (Product # 10006D). Normal Rabbit IgG was used as a negative IP control (Lane 2). 10% input represents the cell extract used for immunoprecipitation (Lane 1). Western blot analysis was performed using Syntaxin 6 Recombinant Rabbit Polyclonal Antibody (Product # 710855) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 æg/mL, 1:2500 dilution). Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).