- MA5-33215 - Invitrogen Antibodies BAK1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-BAK Recombinant Rabbit Monoclonal Antibody (Product # MA5-33215) and a 25 kDa band corresponding to BAK1 was observed across cell lines tested. Membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), Ramos (Lane 2), K-562 (Lane 3) 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 # A27036,1:5000 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)
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Experimental details: Western Blot analysis of precipitated BAK from HEK293 whole cell lysate using a BAK monoclonal antibody (Product # MA5-33215). An HRP-conjugated Protein G antibody was used as the secondary antibody (1:2000). Lane 1: Rabbit control IgG. Lane 2: HEK293 whole cell lysate (500µg). Lane 3: HEK293 whole cell lysate (20µg).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of BAK1 was achieved by transfecting MCF7 with BAK1 specific siRNAs (Silencer® select Product # S1880, S1879). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the BAK1 untransfected cells (lane 1), non-targeting scrambled siRNA transfected cells (lane 2) and knockdown cells (lane 3). The blot was probed with BAK Recombinant Rabbit Monoclonal Antibody (Product # MA5-33215, 1:1000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5000 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 BAK1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of BAK using a BAK Monoclonal antibody (Product # MA5-33215) at a concentration of 0.9 µg/mL. Positive WB detected in: Hela whole cell lysate, MCF-7 whole cell lysate, THP-1 whole cell lysate, 293 whole cell lysate, A549 whole cell lysate, K562 whole cell lysate, Colo320 whole cell lysate. A secondary Goat polyclonal antibody to rabbit IgG was applied at a 1:50,000 dilution. Observed band size: 24 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of BAK1 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.01% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with BAK Recombinant Rabbit Monoclonal Antibody (Product # MA5-33215) at 1:100 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 ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing mitochondria and cell membrane 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)
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Experimental details: Immunohistochemical analysis of BAK in paraffin embedded human endometrial cancer using a BAK monoclonal antibody (Product # MA5-33215) at a dilution of 1:90. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated SP system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of BAK in paraffin embedded human lymph node tissue using a BAK monoclonal antibody (Product # MA5-33215) at a dilution of 1:90. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated SP system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of BAK in Hela cells using a monoclonal antibody (Product # MA5-33215) at a dilution of 1:50. The cells were fixed with 70% Ethyl alcohol (18h) and then permeabilized with 0.3% Triton X-100 for 2 min. The cells were then incubated in 1x PBS /10% normal goat serum to block non-specific protein-protein interactions followed by primary antibody for 1 h at 4°C. The secondary antibody used was FITC goat anti-rabbit IgG (H+L) at 1:200 dilution for 1 h at 4°C. Control antibody (green line) was used under the same conditions. Acquisition of >10,000 events was performed.

MA5-33215