Anti-HSF1 antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HSF1 antibody (AB2923)

Immunocytochemistry/Immunofluorescence analysis of HSF1 (green) in HeLa (Human epithelial cell line from cervix adenocarcinoma) and NIH/3T3 (Mouse embryo fibroblast cell line) cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with ab2923 (1 : 50) for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat-anti rabbit IgG secondary antibody (1 : 400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

• IP

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Immunoprecipitation - Anti-HSF1 antibody (AB2923)

Immunoprecipitation of HSF1 was performed on HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. Antigen : antibody complexes were formed by incubating 500μg whole cell lysate with 2μg of ab2923 overnight on a rocking platform at 4°C. Immune complexes were captured on 50μl Protein A/G Plus Agarose, washed extensively, and eluted with buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 (1 : 1000) overnight rotating at 4°C, washed in TBST, and probed with detection reagent (1 : 1000) for at least one hour. Chemiluminescent detection was performed.

All lanes:

Immunoprecipitation - Anti-HSF1 antibody (ab2923)

Predicted band size: 57 kDa

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• WB

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Western blot - Anti-HSF1 antibody (AB2923)

Western blot analysis of Heat Shock Factor 1 (HSF1) was performed by loading samples onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 overnight at 4°C on a rocking platform, washed in TBS-0.1% Tween 20, and probed with a secondary antibody for at least one hour. Chemiluminescent detection was performed.

All lanes:

Western blot - Anti-HSF1 antibody (ab2923) at 1/1000 dilution

Lane 1:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 50 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate at 50 µg

Lane 4:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 50 µg

Lane 5:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 50 µg

Lane 6:

COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate at 50 µg

Lane 7:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 50 µg

Lane 8:

NRK (Rat kidney normal tissue) whole cell lysate at 50 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG secondary antibody at 1/20000 dilution

Predicted band size: 57 kDa

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• WB

Supplier Data

Western blot - Anti-HSF1 antibody (AB2923)

Western blot analysis of HSF1 was performed by loading each sample onto an SDS-PAGE gel. Proteins were transferred to PVDF membrane and followed by blocking in TBST+5% non-fat milk. HSF1 was detected using the primary anitbody (ab2923) in TBST+2% non-fat milk overnight at 4°C on a rocking platform, followed by secondary antibody for 30 min. Detection was performed using and 1-Step™ TMB-Blotting Substrate Solution.

All lanes:

Western blot - Anti-HSF1 antibody (ab2923) at 1/5000 dilution

Lane 1:

HSF1 RNAi treated C. elegans whole worm tissue at 10 µg

Lane 2:

Untreated C. elegans whole worm tissue at 10 µg

Secondary

All lanes:

anti-rabbit IgG HRP-linked at 1/5000 dilution

Predicted band size: 57 kDa

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