- PA1-852 - Invitrogen Antibodies TRIM28 antibody

Matsuda E, Agata Y, Sugai M, Katakai T, Gonda H, Shimizu A
The Journal of biological chemistry 2001 Apr 27;276(17):14222-9

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot on recombinant human KAP-1 using PA1-852.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TRIM28 was performed by loading 10 g of whole cell human BJ fibroblast protein lysate and run on a 4-12% BTE gel. Proteins were transferred to PVDF membrane. Membrane was blocked in 5% non-fat milk in TBST. TRIM28 was detected at approximately 90 kDa using a TRIM28 polyclonal antibody (Product # PA1-852) at a dilution of 1:250 (2 µg/mL) in 5% milk, followed by a 1:10,000 dilution of anti-rabbit HRP. Chemiluminescent detection was performed using SuperSignal West Pico PLUS substrate (Product # 34580). Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of TRIM28 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR801077_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of TRIM28 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2) and HeLa TRIM28 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-TRIM28 Polyclonal Antibody (Product # PA1-852) using 1:3,000 dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to TRIM28. An uncharacterized band was observed at ~60 kDa in all the samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of KAP1 (green) showing staining in the nucleus of L6 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a KAP1 polyclonal antibody (Product # PA1-852) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor 554 (red) and nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of KAP1 (green) showing staining in the nucleus of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a KAP1 polyclonal antibody (Product # PA1-852) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor 554 (red) and nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of KAP1 showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a KAP1 polyclonal antibody (Product # PA1-852) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of KAP1 showing staining in the nucleus of paraffin-embedded mouse spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a KAP1 polyclonal antibody (Product # PA1-852) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of KAP1 showing staining in the nucleus of paraffin-embedded rat spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a KAP1 polyclonal antibody (Product # PA1-852) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

PA1-852