Anti-PIAS1 antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PIAS1 antibody (AB32219)

ICC/IF image of ab32219 stained MEF1 cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab32219, 1μg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

• WB

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Western blot - Anti-PIAS1 antibody (AB32219)

ab32219 was used in western blot analysis using HeLa whole cell lysate, HeLa nuclear lysate and T47D nuclear lysate. It was unable to detect endogenous PIAS1 in these lysates. It was able to detect PIAS1 in cells that are overexpressing the protein, running at a molecular weight of approximately 81kDa, suggesting that the amount of endogenous PIAS1 in these cells is very low. When ab32219 was used to immunoprecipitate the PIAS1 from these extracts, the protein could then be detected by western blot in the overexpressing HeLa extract as well as the T47D nuclear extract.For immunoprecipitations, 25ug of extract was used in the IP, and 10% of the sample was run on the gel.

All lanes:

Western blot - Anti-PIAS1 antibody (ab32219) at 1/1000 dilution

Lane 1:

HeLa whole cell lysate at 25 µg

Lane 2:

HeLa whole cell extract with transfected PIAS1 at 25 µg

Lane 3:

HeLa nuclear extract at 25 µg

Lane 4:

T47D nuclear extract at 25 µg

Lane 5:

HeLa whole cell lysate immunoprecipitated with ab32219

Lane 6:

HeLa whole cell extract with transfected PIAS1 immunoprecipitated with ab32219

Lane 7:

HeLa nuclear extract immunoprecipitated with ab32219

Lane 8:

T47D nuclear extract immunoprecipitated with ab32219

Predicted band size: 71 kDa

Observed band size: 110 kDa,79 kDa,81 kDa

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• WB

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Western blot - Anti-PIAS1 antibody (AB32219)

All lanes:

Western blot - Anti-PIAS1 antibody (ab32219) at 1 µg/mL

All lanes:

Human testis tissue lysate - total protein (<a href='/en-us/products/unavailable/human-testis-tissue-lysate-total-protein-ab30257'>ab30257</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Predicted band size: 71 kDa

Observed band size: 180 kDa,72 kDa

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Exposure time: 4min

• WB

Project

Western blot - Anti-PIAS1 antibody (AB32219)

All lanes:

Western blot - Anti-PIAS1 antibody (ab32219) at 1 µg/mL

All lanes:

PIAS1 - Recombinant Protein at 0.1 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 71 kDa

Observed band size: 100 kDa

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Exposure time: 2min

• WB

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Western blot - Anti-PIAS1 antibody (AB32219)

All lanes:

Western blot - Anti-PIAS1 antibody (ab32219) at 1/2500 dilution

Lane 1:

293T cell lysate at 25 µg

Lane 2:

MEL cell lysate at 25 µg

Lane 3:

K562 cell lysate at 25 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/donkey-rabbit-igg-h-l-hrp-ab6802'>ab6802</a>) at 1/20000 dilution

Predicted band size: 71 kDa

Observed band size: 71 kDa

true

Exposure time: 1min

This image is courtesy of an Abreview submitted by Min young Kim