- MA5-14815 - Invitrogen Antibodies PKM antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PKM1/2 was performed by loading 30 µg of THP-1 whole cell lysate per well onto an SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature. The membrane was probed with a PKM1/2 monoclonal antibody (Product # MA5-14815) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:40,000 for 1 hour at room temperature. Chemiluminescent detection was performed using ECL substrate. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofPKM (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR733142_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of PKM was performed by loading 30 µg of HeLa Cas9 (Lane 1) and HeLa Cas9 PKM Lentiviral sgRNA (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 PKM1/PKM2 Monoclonal Antibody (F.313.7) (Product # MA5-14815, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toPKM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PKM1/2 in extracts from MCF-7 and RD cells using PKM1/2 monoclonal antibody (Product # MA5-14815).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of PKM1 was achieved by transfecting MDA-MB-231 cells with PKM1 specific siRNAs (Silencer® select Product # S10575, S10574). Western blot analysis (Fig. a) was performed using Whole cell extracts from the PKM1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with PKM1 Monoclonal Antibody (F.313.7) (Product # MA5-14815,1:1000 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). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PKM1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PKM1 Monoclonal Antibody (F.313.7)(Product # MA5-14815) and a 58kDa band corresponding to PKM1 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), HEK-293 (Lane 2), Hep G2 (Lane 3) and HeLa (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PKM1 was performed using 70% confluent log phase MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with PKM1 Monoclonal Antibody (F.313.7) (Product # MA5-14815) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic 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: Immunofluorescent analysis of PKM1/2 (red) in HEK293T cells. Cells fixed with 4% formaldehyde were permeabilized and blocked with 1X PBS containing 5% BSA and 0.3% Triton X-100 for 1 hour at room temperature. Cells were probed with a PKM1/2 monoclonal antibody (Product # MA5-14815) at a dilution of 1:100 overnight at 4°C in 1X PBS containing 1% BSA and 0.3% Triton X-100, washed with 1X PBS, and incubated with a fluorophore-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:200 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI. Images were taken on a Leica DM1000 microscope at 40X magnification. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of PKM1/2 was performed on THP-1 cells. Antigen-antibody complexes were formed by incubating 500 µg of THP-1 whole cell lysate (in 500 µL volume) with 5 µL of a PKM1/2 monoclonal antibody (Product # MA5-14815) overnight at 4°C. The immune complexes were captured on 30 µL of protein G agarose, washed extensively, and eluted with 6X Laemmli non-reducing sample buffer. Samples were resolved on an 8% SDS-PAGE gel, transferred to a PVDF membrane, and blocked with 5% milk in TBST for 1 hour at room temperature. The membrane was probed with a PKM1/2 monoclonal antibody (Product # MA5-14815) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:40,000 for 1 hour at room temperature. Chemiluminescent detection was performed using ECL substrate. Data courtesy of the Innovators Program.

MA5-14815