Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Immunohistochemistry [2]
- Flow cytometry [1]
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Validation data
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- Product number
- MA5-33215 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- BAK Recombinant Rabbit Monoclonal Antibody (8D1)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 8D1
- Vial size
- 100 µL
- Concentration
- 0.6 mg/mL
- Storage
- -20°C or -80°C if preferred
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Supportive validation
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- 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).
- 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).
- 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.
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- 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
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- 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
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- Invitrogen Antibodies (provider)
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- 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.
- 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.