All lanes : Anti-PIAS1 antibody (ab32219) at 1/2500 dilution (Incubation for 1 hour at 25°C.)

Lane 1 : 293T cell lysate
Lane 2 : MEL cell lysate
Lane 3 : K562 cell lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab6802) at 1/20000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 71 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

See Abreview

All lanes : 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


Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 81 kDa why is the actual band size different from the predicted?
Additional bands at: 110 kDa, 79 kDa (possible cleavage fragment), 79 kDa (possible cross reactivity). We are unsure as to the identity of these extra bands.



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.

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).

Anti-PIAS1 antibody (ab32219) at 1 µg/ml + PIAS1 - Recombinant Protein at 0.1 µg

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

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

ab32219 recognizes the full length tagged recombinant protein which has an expected molecular weight of 98 kDa.

Anti-PIAS1 antibody (ab32219) at 1 µg/ml + Human testis tissue lysate - total protein (ab30257) at 10 µg

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 72 kDa
Additional bands at: 180 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes