PA5-84414

antibody from Invitrogen Antibodies

Targeting: PBRM1 BAF180, PB1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-84414

Provider

Invitrogen Antibodies

Product name

PBRM1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant full-length protein

Description

Immunogen sequence: PVIQQPTTPM FVAPPPKTQR LLHSEAYLKY IEGLSAESNS ISKWDQTLAA RRRDVHLSKE QESRLPSHWL KSKGAHTTMA DALWRLRDLM LRDTLNIR

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

0.2 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PBRM1 by a PBRM1 polyclonal antibody (Product # PA5-84414). Analysis in human cell line RT-4.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of PBRM1 was achieved by transfecting HeLa with PBRM1 specific siRNAs (Silencer® select Product # S30402, S30400). Western blot analysis (Fig. a) was performed using fresh nuclear enriched extracts from the PBRM1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with PBRM1 Polyclonal Antibody (Product # PA5-84414, 1:200 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dlution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PBRM1.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using PBRM1 Polyclonal Antibody (Product # PA5-84414) and a 200 kDa band corresponding to PBRM1 was observed across cell lines tested. Nuclear enriched extracts (40 µg lysate) of ACHN (Lane 1), 769-P (Lane 2), PC-3 (Lane 3), DU 145 (Lane 4), Jurkat (Lane 5), HEK-293 (Lane 6), IMR-32 (Lane 7), HeLa (Lane 8) and MCF7 (Lane 9) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA03752BOX), 12 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:200 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using PBRM1 Polyclonal Antibody (Product # PA5-84414) and a 200 kDa band corresponding to PBRM1 was observed across cell lines tested. Fresh nuclear enriched extracts (40 µg lysate) of Jurkat (Lane 1), HeLa (Lane 2) and MCF7 (Lane 3) prepared using RIPA lysis buffer supplemented with Universal Nuclease for Cell Lysis were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA03752BOX), 12 well. Resolved proteins were equilibrated with 20% ethanol and then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:200 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of PBRM1 was performed using 70% confluent log phase HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PBRM1 Polyclonal Antibody (Product # PA5-84414) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing nuclear as well as cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of PBRM1 was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PBRM1 Polyclonal Antibody (Product # PA5-84414) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing nuclear as well as cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of PBRM1 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PBRM1 Polyclonal Antibody (Product # PA5-84414) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing nuclear as well as cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of PBRM1 in RT4 cells using a PBRM1 polyclonal antibody (Product # PA5-84414). The analysis shows localization to nucleoplasm.