701494

antibody from Invitrogen Antibodies

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

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

701494

Provider

Invitrogen Antibodies

Product name

c-Kit Recombinant Rabbit Monoclonal Antibody (HC34LC14)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with bovine, equine, goat, non-human primate, rat and porcine based on sequence homology.

Antibody clone number

HC34LC14

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of c-Kit/CD117 was performed by loading 20 µg of HeLa (lane1), Jurkat (lane2) and K562 (lane3) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. c-Kit/CD117 was detected at ~80 kDa using c-Kit/CD117 Recombinant Rabbit Monoclonal Antibody (Product # 701494) at 0.5-1 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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 analysis of c-Kit in whole cell extracts of HeLa, Jurkat, and K562 cells (lanes 1-3 respectively) using a c-Kit recombinant rabbit monoclonal antibody (Product # 701494) at a dilution of 1 µg/mL. Detection was performed using an HRP-conjugated goat anti-rabbit secondary antibody followed by chemiluminescence (ECL). Results show a band at ~80kDa.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of C- Kit (CD117) was done on 70% confluent log phase K562 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with C- Kit (CD117) Recombinant Rabbit Monoclonal Antibody (Product # 701494) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c is a merged image showing cytoplasmic localization of C- Kit (CD117). The images were captured at 20X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of C-kit/CD117 was done on Jurkat cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª C-kit/CD117 Recombinant Rabbit Monoclonal Antibody (701494, red histogram) or with rabbit isotype control (pink histogram) at 1-3 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.