- PA5-27215 - Invitrogen Antibodies RB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Rb using 30 µg A431 whole cell lysate. Samples were loaded onto a 5% SDS-PAGE gel and probed with a Rb polyclonal antibody (Product # PA5-27215) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of Rb was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a Rb Polyclonal Antibody (Product # PA5-27215) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using Rb Polyclonal Antibody (Product # PA5-27215). Various whole cell extracts (30 µg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Rb Polyclonal Antibody (Product # PA5-27215) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Rb was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1008282_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Rb was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) andHeLa Rb KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Rb Polyclonal Antibody (Product # PA5-27215, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:8000 dilution) using the iBright™ FL 1500 (Product #A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Rb.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Retinoblastoma-associated protein was achieved by transfecting MCF7 with Retinoblastoma-associated protein specific siRNAs (Silencer® select Product # s524, s523). Western blot analysis (Fig. a) was performed using Modified whole cell extracts (1% SDS) (30 µg lysate) from the Retinoblastoma-associated protein knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Rb Polyclonal Antibody (Product # PA5-27215, 1:1000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Retinoblastoma-associated protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Rb Polyclonal Antibody (Product # PA5-27215) and a 110 kDa band corresponding to Retinoblastoma-associated protein was observed across the cell lines and tissues tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of MCF7 (Lane 1), LNCaP (Lane 2), HeLa (Lane 3), A-431 (Lane 4), SW480 (Lane 5) and tissue extracts (30 µg lysate) of Mouse Retina (Lane 6) and Rat Retina (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band of ~70 kDa was also observed in LNCaP, ~50 kDa band in A431 and ~32 kDa bands in LNCaP, A431 and SW480.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of Rb was performed in MCF7 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: Rb Polyclonal Antibody (Product # PA5-27215) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Rb Polyclonal Antibody detects Rb protein at nucleus by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: Rb stained by Rb Polyclonal Antibody (Product # PA5-27215) diluted at 1:500. Red: phalloidin, a cytoskeleton marker, diluted at 1:200. Scale bar= 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Retinoblastoma-associated protein was performed using 70% confluent log phase MCF7 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 Rb Polyclonal Antibody (Product # PA5-27215) 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 ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Rb Polyclonal Antibody detects Rb protein at nucleus by immunohistochemical analysis. Sample: Paraffin-embedded mouse eye. Green: Rb stained by Rb Polyclonal Antibody (Product # PA5-27215) diluted at 1:500. Red: beta Tubulin 3/ Tuj1, a cytoskeleton marker, stained by beta Tubulin 3/ Tuj1 antibody [GT11710] diluted at 1:500. Blue: Fluoroshield with DAPI. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Rb Polyclonal Antibody detects Rb protein at nucleus on human colon carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human colon carcinoma. Rb Polyclonal Antibody (Product # PA5-27215) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Rb Polyclonal Antibody immunoprecipitates retinoblastoma 1 protein in IP experiments. IP samples: HeLa whole cell extract. A. Control with 4 µg of preimmune Rabbit IgG. B. Immunoprecipitation of retinoblastoma 1 protein by 4 µg Rb Polyclonal Antibody (Product # PA5-27215). 7.5 % SDS-PAGE. The immunoprecipitated retinoblastoma 1 protein was detected by Rb Polyclonal Antibody (Product # PA5-27215) diluted at 1:500.

PA5-27215

Antibody data