重组Anti-PIAS1抗体[EPR2580(2)]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

Immunohistochemical analysis of paraffin-embedded Human colon tissue using 1/100 ab109388.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling PIAS1 with purified ab109388 at 1/500 dilution (4.6 μg/mL). Cells were fixed in 100% Methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain.

• WB

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Western blot - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

All lanes:

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (ab109388) at 1/1000 dilution

Lane 1:

HepG2 cell lysates at 10 µg

Lane 2:

Jurkat cell lysates at 10 µg

Lane 3:

HEK-293 cell lysates at 10 µg

Lane 4:

Daudi cell lysates at 10 µg

Predicted band size: 71 kDa

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• WB

Lab

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

Exposure time :

Lane 1 : 10 seconds.

Lane 2 : 1 minute.

Blocking and dilution buffer : 5% NFDM /TBST.

Lane 1:

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (ab109388) at 1/500 dilution

Lane 2:

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (ab109388) at 1/2000 dilution

All lanes:

Recombinant Human PIAS1 protein (<a href='/products/unavailable/recombinant-human-pias1-protein-ab152888'>ab152888</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 71 kDa

Observed band size: 98 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

Western Blotting using Anti-PIAS1 antibody [EPR2580(2)], ab109388. Publication image from Chiou, H. Y. et al., 2014, J Biomed Sci, 24894488. Legend direct from paper.

PIAS1 enhances the suppressing effect of Hes-1 on GADD45α expression that is blocked by Hes-1 3KR. Different doses of Flag-PIAS1WT or Flag-PIAS1W372A plasmid was co-transfected with Myc-SUMO-1 and various doses of Flag-Hes-1WT or Flag-Hes-1 3KR plasmid together with 0.4 µg of GADD45α promoter construct to HEK293T cells for determination of (A) GADD45α promoter activity and (B) GADD45α mRNA level by real-time PCR. (C) Different doses of Flag-PIAS1WT or Flag-PIAS1W372A plasmid was co-transfected with various doses of Flag-Hes-1WT or Flag-Hes-1 3KR plasmid to HEK293T cells and western blot against GADD45α and Flag was performed. The quantified result is shown on the right panel. (D) Two sets of PIAS1 siRNA and 0.4 µg of GADD45α promoter construct were transfected to HEK293T cells and GADD45α promoter activity was determined 48 h later. Two sets of PIAS1 siRNA were transfected to HEK293T cells and (E) GADD45α mRNA level and (F) GADD45α protein level were determined 48 h later. The effectiveness of PIAS1 siRNA transfection is confirmed by western blot against PIAS1. (G) Two doses of Flag-GADD45α plasmid was transfected to cells with the addition of different concentrations of H2O2 and cell apoptosis was determined by TUNEL assay. Western blot against the Flag-tag confirms plasmid transfection and expression. (H) Two concentrations of GADD45α siRNA was transfected to HEK293T cells with the addition of different concentrations of H2O2 and cell apoptosis was determined by TUNEL assay. Western blot against GADD45α confirms the effectiveness of GADD45α siRNA transfection. Data are expressed as that in Figure 6. Each experiment was performed twice. *p < 0.05, **p < 0.01 and # p < 0.001.

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• WB

CiteAb

Western blot - Anti-PIAS1 antibody [EPR2580(2)] (AB109388)

Western Blotting using Anti-PIAS1 antibody [EPR2580(2)], ab109388. Publication image from Chiou, H. Y. et al., 2014, J Biomed Sci, 24894488. Legend direct from paper.

H2O2 enhances the SUMOylation of Hes-1. (A) Flag-Hes-1WT plasmid was transfected to HEK293T cells with the addition of different concentrations of H2O2 and the cell lysate was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Hes-1 (left) or anti-SUMO-1 (right) antibody. (B) Flag-Hes-1WT plasmid was transfected to HEK293T cells with the addition of 0.2 mM H2O2 for different time periods. The cell lysate was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Hes-1 (left) or anti-SUMO-1 (right) antibody. (C) Myc-SUMO-1 plasmid was transfected to HEK293T cells with the addition of 0.2 mM H2O2 for 4 h. The cell lysate was immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Hes-1 and anti-SUMO-1 antibody. (D) H2O2 (0.2 mM) was added to HEK293T cells for different time periods and the cell lysate was subject to western blot against pSer-90 PIAS1 and PIAS1 (upper panel). Flag-Hes-1WT plasmid was transfected to HEK293T cells with co-transfection of EGFP-PIAS1WT or EGFP-PIAS1S90A plasmid and with the addition of H2O2 for 4 h and the cell lysate was immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies against PIAS1 and Flag. Western blot against EGFP and Flag for cell lysates only was used as a control for transfection and expression (lower panel). (E) Flag-Hes-1WT plasmid was co-transfected with EGFP-PIAS1 and Myc-SUMO-1 plasmids, and H2O2 was added to some of these groups for 4 h. The phosphatase inhibitor CIP (20 U/ml) was also added to some of these groups. The cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-PIAS1 antibody. Western blot against Flag, PIAS1 and SUMO-1 for cell lysates only was used as loading controls. The sumoylated Hes-1 bands are shown in the bracket. Each experiment was performed twice.

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