MA3-025

antibody from Invitrogen Antibodies

Targeting: CCNH CycH, p34, p37

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Data presented on provider website)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA3-025

Provider

Invitrogen Antibodies

Product name

Cyclin H Monoclonal Antibody (33TF2-2D4)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

MA3-025 has been used successfully in Western blotting, Immunoprecipitation and Immunofluorescence applications to detect Cyclin H in human samples.

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

33TF2-2D4

Vial size

50 µL

Concentration

Conc. Not Determined

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Cyclin H was performed by loading 50 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a Novex 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a PVDF membrane using the G2 Blotter (Product # 62288), and blocked with Starting Block T20 (Product # 37543) for 1 hour at room temperature. Cyclin H was detected at ~36 kD using a Cyclin H monoclonal antibody (Product # MA3-025) at a dilution of 1:2000 in blocking buffer for 1 hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Cyclin H (green) in U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were stained with a Cyclin H monoclonal antibody (Product # MA3-025) at a dilution of 1:200 in staining buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG Secondary Antibody, DyLight 488 conjugate (Product # 35502) at a dilution of 1:500 for 1 hour at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of Cyclin H was performed on K562 cells. Antigen-antibody complexes were formed by incubating 250 µg of K562 whole cell lysate with 1 µg of a Cyclin H monoclonal antibody (Product # MA3-025) overnight on a rocking platform at 4C. The immune complexes were captured on 50 µL of A/G Plus Agarose (Product # 20423), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). K562 whole cell lysate (25 µg) was loaded as a positive control (left lane). Samples were resolved on a Novex 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ), transferred to a PVDF membrane, and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. Cyclin H was detected at ~36 kD using a Cyclin H monoclonal antibody (Product # MA3-025) at a dilution of 1:2000 in blocking buffer for 1 hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:20,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).