14-5069-82

antibody from Invitrogen Antibodies

Targeting: ASH2L ASH2, ASH2L1, ASH2L2, Bre2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

14-5069-82

Provider

Invitrogen Antibodies

Product name

ASH2L Monoclonal Antibody (AS 4C5), eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

This AS 4C5 monoclonal antibody recognizes human ASH2L, which has been described as a transcriptional regulator and may play a role in hematopoiesis. AS 4C5 detects full length (unspliced) and reduced ASH2L by Western Blot and stains ASH2L in the nucleoplasm and at the plasma membrane in Immunocytochemistry. ASH2L monoclonal antibody (AS 4C5) has been validated for the following applications: Western Blot and Immunocytochemistry; AS 4C5 does not cross react with mouse ASH2L. Applications Reported: This AS 4C5 antibody has been reported for use in Western Blot and Immunocytochemistry. Applications Tested: This AS 4C5 antibody has been tested by western blot using whole cell lysates of HeLa cells (human) and NIH 3T3 or N2a (mouse cell lines) and by immunocytochemistry on HeLa cells. This AS 4C5 antibody may be used at 2 µg/mL for western blot and at 1.25 µg/mL for immunocytochemistry.

Reactivity

Human

Host

Rat

Isotype

IgG

Antibody clone number

AS 4C5

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

4° C, do not freeze

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis was performed using whole cell extracts of HeLa (Lane 2) and NIH 3T3 cells (Lane 3) on a 4-12% Nupage Bis-Tris Gel (Product # NP0321BOX) for gel electrophoresis in MOPS SDS Running Buffer (Product # NP0001). Protein transfer to a nitrocellulose membrane was achieved using the iBlot 2 system. Consecutively, the membrane was probed with ASH2L Monoclonal Antibody (AS 4C5) (Product # 14-5069-82) (2 µg/mL) and detected by chemiluminescence using Goat-anti Rat IgG Secondary Antibody, HRP (Product # 31470) at 1:10.000 dilution. A 70 kDa band was detected in HeLa (human) lysate but not in NIH 3T3 (mouse) lysate using Super Signal West Pico chemiluminescence substrate. Protein size was determined using SeeBlue Plus2 Pre-stained Protein Standard (Product # LC5925) (Lane 1).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis was performed using whole cell extracts of HeLa (Lane 2) and NIH 3T3 cells (Lane 3) on a 4-12% Nupage Bis-Tris Gel (Product # NP0321BOX) for gel electrophoresis in MOPS SDS Running Buffer (Product # NP0001). Protein transfer to a nitrocellulose membrane was achieved using the iBlot 2 system. Consecutively, the membrane was probed with ASH2L Monoclonal Antibody (AS 4C5) (Product # 14-5069-82) (2 µg/mL) and detected by chemiluminescence using Goat-anti Rat IgG Secondary Antibody, HRP (Product # 31470) at 1:10.000 dilution. A 70 kDa band was detected in HeLa (human) lysate but not in NIH 3T3 (mouse) lysate using Super Signal West Pico chemiluminescence substrate. Protein size was determined using SeeBlue Plus2 Pre-stained Protein Standard (Product # LC5925) (Lane 1).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of ASH2L (green) in HeLa cells (65% confluency). The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 1% Triton X-100 for 30 minutes at 37 C, and blocked with 10% Normal Goat Serum in PBS with 0.1% Triton X-100 for 1 hour at room temperature. Cells were stained overnight with the ASH2L mouse monoclonal antibody (Product # 14-5069-82) at 4 C at a concentration of 1.25 µg/mL in blocking buffer followed by a Goat-anti Rat IgG Secondary Antibody, Alexa Fluor 488 (Product # A-11006) at a dilution of 4 µg/mL in blocking buffer for 1 hour at room temperature. Nuclei (blue) were stained with DAPI in Fluoromount G with DAPI (Product # 00-4959-52). Images were taken on a Zeiss Axio Observer.Z1 microscope at 20 x magnification with an Orca-Flash4.0. camera from Hamamatsu. The upper panel shows cells stained with clone AS 4C5. No non-specific staining was observed with rat IgG2a isotype control (lower panel).