710762

antibody from Invitrogen Antibodies

Targeting: KIT C-Kit, CD117, PBT, SCFR

Antibody data

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Validation data

Product number: 710762 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-c-Kit (Tyr703) Recombinant Polyclonal Antibody (22HCLC)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody is predicted to react with Monkey, Rat, Mouse, Pig, Bovine, Rabbit and Dog.
Antibody clone number: 22HCLC
Concentration: 0.5 mg/mL

KIT protein structure - 710762 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Jurkat (Lane 1), A549 (Lane 2), SH-SY5Y (Lane 3), HeLa (Lane 4). The blots were probed with Anti-c-kit (pY703) Recombinant Rabbit Polyclonal Antibody (Product # 710762, 1-3 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 50 kDa band corresponding to an isoform of ckit (pY703) was observed across cell lines tested. B) To confirm the specificity of Anti-c-kit (pY703) Recombinant Rabbit Polyclonal Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the corresponding blot on the right. The peptide competes with the antibody and prevents it from binding to the membrane. 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 by overnight wet transfer method. 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Phospho-c-Kit (Tyr703) Recombinant Polyclonal Antibody (22HCLC) (Product # 710762) and a 55 kDa band corresponding to Mast/stem cell growth factor receptor Kit was observed across cell lines tested. Whole cell extracts (30 µg lysate) of IMR-32 (Lane 1), IMR-32 treated with SCF (100 ng/mL, 30 min) (Lane 2), SH-SY5Y (Lane 3), SH-SY5Y treated with SCF (100 ng/mL, 30 min) (Lane 4), HeLa (Lane 5), HeLa treated with SCF (100 ng/mL, 30 min) (Lane 6),K-562 (Lane 7) and HEL 92.1.7 (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 µg/mL) 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: Immunofluorescence was performed on fixed and permeabilized A549 cells for detection of c-kit (pY703) using Anti-c-kit (pY703) Recombinant Rabbit Polyclonal Antibody (Product # 710762, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of c-kit (pY703) (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane and perinuclear localization of c-kit (pY703). Panel e) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Mast/stem cell growth factor receptor Kit was performed using 70% confluent log phase HT-29,SCF, 100 ng/mL, 30 min. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Phospho-c-Kit (Tyr703) Recombinant Polyclonal Antibody (22HCLC) (Product # 710762) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing 60x localization. Panel e represents no expression of phospho c-KIT. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.