437000
antibody from Invitrogen Antibodies
Targeting: GAPDHS
GAPD2, GAPDH-2, GAPDS
Antibody data
- Antibody Data
- Antigen structure
- References [0]
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Immunohistochemistry [2]
- Flow cytometry [1]
- Other assay [9]
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- Product number
- 437000 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- GAPDH Monoclonal Antibody (258)
- Antibody type
- Monoclonal
- Antigen
- Recombinant full-length protein
- Reactivity
- Human, Bovine, Feline, Porcine
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- 258
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- -20°C
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Supportive validation
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- Western blot analysis of GAPDH was performed by loading 20 µg of SH-SY5Y (lane1), A431 (lane2), K562 (lane3), HeLa (lane4), U-87 MG (lane5) and MCF7 (lane6) cell lysate using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. GAPDH was detected at ~37 kDa using GAPDH Mouse Monoclonal Antibody (Product # 437000) at 1-2 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
Supportive validation
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- Immunofluorescent analysis of GAPDH was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with GAPDH Mouse Monoclonal Antibody (Product # 437000) at 1 µg/mL and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
Supportive validation
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- Immunohistochemistry analysis of GAPDH showing staining in the cytoplasm and nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a GAPDH monoclonal antibody (Product # 437000) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
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- Immunohistochemistry analysis of GAPDH showing staining in the cytoplasm and nucleus of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a GAPDH monoclonal antibody (Product # 437000) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
Supportive validation
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- Flow cytometry analysis of GAPDH was done on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with GAPDH Mouse Monoclonal Antibody (437000, red histogram) or with mouse isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor¨ 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.
Supportive validation
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- Figure 1 Drp1 expression is increased after inhibiting autophagy. (A) HEK-293T cells were incubated with DMSO, Bafilomycin (100 nM), or MG132 (25 muM) for 4 hours and 24 hours and whole cell lysate was analyzed by Western blotting using the indicated antibodies. LC3 levels confirm the bafilomycin is inhibiting autophagic turnover and total ubiquitin levels confirm MG132 effect. (B) HEK-293T cells were incubated with chloroquine (10 muM) for 4 hours and 24 hours followed by Western blotting (C) Lysosomal protease inhibitors E64D (10 mug/ml) and pepstatin (10 mug/ml) were incubated with HEK-293T cells for 24 hours. (D) SH-SY5Y cells were incubated with DMSO, bafilomycin (100 nM), or MG132 (25 muM) for 24 hours.
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- Figure 2 Bafilomycin and ATG7 knockdown increases mitochondrial Drp1. (A) Cytosolic and mitochondrial fractions from bafilomycin treated HEK-293T cells were isolated by differential centrifugation and analyzed by immunoblotting. *Drp1 antibody reactive band that is only seen in the mitochondrial fraction. Cox IV is used as a mitochondrial loading control and Actin indicates most of the cytosol is absent from the mitochondrial fraction. (B) ATG7 stable knockdown clones were created and whole cell lysate was subject to immunoblotting. (C) Mitochondrial fractions of ATG7 knockdown clones were isolated and lysates were probed with the indicated antibodies. VDAC is used as a mitochondrial loading control.
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- FIGURE 3: Silencing of Nck disrupts the organization of F-actin and the distribution of VE-cadherin in cells cultured in 3D collagen matrices. (A) Confocal images showing F-actin (red), endogenous VE-cadherin (green), and nuclei (blue). Lumens (L) are indicated. The ROIs (dotted squares) were magnified and are shown to the right. (B) Surface plots showing pixel intensities for field images in A. (C) Actin and VE-cadherin accumulation at cell-cell contacts. Mean fluorescence intensity was quantified in 23-50 equal-size ROIs pooled from three independent experiments. (D) Quantitation of VE-cadherin cell-cell junctional lengths (top). Data represent mean junctional length (mum) determined in a total of 25-30 junctions pooled from three individual experiments. The Western blots (bottom) show the levels of VE-cadherin, Nck, and GAPDH (loading control) in cell extracts from 3D cultures.
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- FIGURE 5: Loss of Nck disrupts activation of endogenous Cdc42 in cells cultured in 3D collagen matrices. (A) Confocal images showing the cytoskeletal architecture (F-actin; red), distribution of GTP-bound Cdc42 (Cdc42 GTP ; green), and nuclei (DNA; blue). Lumens (L) are indicated. The ROIs (dotted squares) were magnified and are shown to the right. Scale bar, 15 mum. (B) Line scans showing intensities along the solid white lines displayed in A. (C) Quantification of luminal Cdc42 fluorescence intensity. Average pixel intensity was extracted from 35-50 ROIs from images acquired in three independent experiments. (D) Representative Western blots showing levels of GTP-bound Cdc42 (PBD Pak1 pull-down assays), total Cdc42, Nck, and GAPDH in cell extracts obtained from 3D cultures. (E) Quantification of active Cdc42. The intensity of bands corresponding to active Cdc42 (PBD Pak1 pull-down assays) was normalized by the intensity of bands corresponding to total Cdc42 in cell extracts and expressed as percentage of control cultures ( n = 3 independent experiments).
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- FIGURE 7: Loss of Nck disrupts the activation of endogenous aPKC in cells cultured in 3D collagen matrices. (A) Confocal images showing the cytoskeletal architecture (F-actin; red), distribution of active aPKC (p-aPKC; green), and nuclei (DNA; blue). Lumens (L) are indicated. The ROIs (dotted squares) were magnified and are shown to the right. (B) Line scans showing intensities along the solid white lines displayed in A. (C) Quantification of luminal p-aPKC fluorescence intensity. Average pixel intensity was extracted from 35-50 ROIs from images acquired in three independent experiments. (D) Representative Western blots showing levels of p-aPKC, total aPKC, Nck, and GAPDH in cell extracts from 3D cultures. (E) Quantification of active aPKC. The intensity of bands corresponding to p-aPKC was normalized by the intensity of bands corresponding to total aPKC and expressed as percentage of control cultures ( n = 3 independent experiments). a, b, p < 0.05.
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- Figure 5 Abemaciclib causes increased PARP cleavage in RCC In 786-O cells (A) and Caki-1 cells (B) abemaciclib exposure results in increased PARP cleavage. This effect is more rapid and pronounced when abemaciclib is combined with sunitinib.