GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01]
cat.: ET1601-4
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat, Chicken, Zebrafish
Applications: WB, IF-Cell, IF-Tissue, IHC-P, FC, IP
Clonality: Monoclonal
Clone number: SA30-01
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 36 kDa
Isotype: IgG
Immunogen: Recombinant protein within mouse GAPDH aa 94-333 / 333.
Positive control: Rat liver tissue lysate, rat lung tissue lysate, rat heart tissue lysate, rat cerebellum tissue lysate, rat skeletal muscle tissue lysate, rat spleen tissue lysate, rat small intestine tissue lysate, hybrid fish (crucian-carp) brain tissue lysates, PC-3 cell lysate, mouse colon tissue lysate, SH-SY5Y cell lysate, NIH/3T3 cell lysate, SK-Br-3 cell lysate, rat brain tissue lysate, A549, HepG2, human liver tissue, mouse liver tissue, mouse spleen tissue, DF-1 cell lysate.
Subcellular location: Cytoplasm, Nucleus, Membrane
Recommended Dilutions:
  WB
  IF-Cell
  IF-Tissue
  IHC-P
  FC
  IP
1:5,000-1:640,000
1:50
1:50
1:50
1:50
Use at an assay dependent concentration.
Uniprot #: SwissProt: P04406 Human | P16858 Mouse | P04797 Rat
Alternative names: 38 kDa BFA-dependent ADP-ribosylation substrate aging associated gene 9 protein Aging-associated gene 9 protein BARS-38 cb609 EC 1.2.1.12 Epididymis secretory sperm binding protein Li 162eP G3P_HUMAN G3PD G3PDH GAPD GAPDH Glyceraldehyde 3 phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase HEL-S-162eP KNC-NDS6 MGC102544 MGC102546 MGC103190 MGC103191 MGC105239 MGC127711 MGC88685 OCAS, p38 component OCT1 coactivator in S phase, 38-KD component peptidyl cysteine S nitrosylase GAPDH Peptidyl-cysteine S-nitrosylase GAPDH wu:fb33a10
Images
ET1601-4_1.jpg Fig1: Western blot analysis of GAPDH on Hela cell lysates with Rabbit anti-GAPDH antibody (ET1601-4).

Hela cell lysates at 10 µg/Lane.

Predicted band size: 36 kDa
Observed band size: 36 kDa

12% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4) at serial dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
ET1601-4_2.jpg Fig2: Western blot analysis of GAPDH on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4, 1/5,000) was used in 5% NFDM/TBST at room temperature for 1 hour. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 45 mins at room temperature.

Positive control:
Lane 1: PC-3 cell lysate, 10 µg/Lane
Lane 2: Mouse colon tissue lysate, 20 µg/Lane
Lane 3: SH-SY5Y cell lysate, 10 µg/Lane
Lane 4: PC-3 cell lysate, 10 µg/Lane
Lane 5: NIH/3T3 cell lysate, 10 µg/Lane
Lane 6: SK-Br-3 cell lysate, 10 µg/Lane
Lane 7: Rat brain tissue lysate, 20 µg/Lane
ET1601-4_3.jpg Fig3: Western blot analysis of GAPDH on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4, 1/10,000) was used in 5% NFDM/TBST at room temperature for 1 hour. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 45 mins at room temperature.

Positive control:
Lane 1: Rat liver tissue lysate, 20 µg/Lane
Lane 2: Rat lung tissue lysate, 20 µg/Lane
Lane 3: Rat lung tissue lysate, 20 µg/Lane
Lane 4: Rat heart tissue lysate, 20 µg/Lane
Lane 5: Rat cerebellum tissue lysate, 20 µg/Lane
Lane 6: Rat skeletal muscle tissue lysate, 20 µg/Lane
Lane 7: Rat spleen tissue lysate, 20 µg/Lane
Lane 8: Rat small intestine tissue lysate, 20 µg/Lane
ET1601-4_4.jpg Fig4: Western blot analysis of GAPDH on different lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/80,000 dilution.

Cell lysates at 10 µg/Lane, tissue lysates at 20 µg/Lane.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Exposure time: 1 minute;

12% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4) at 1/80,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
ET1601-4_5.jpg Fig5: Western blot analysis of GAPDH on DF-1 cell lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/10,000 dilution.

Cell lysates at 15 µg/Lane.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Exposure time: 1 second;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 2 hour at room temperature. The primary antibody (ET1601-4) at 1/10,000 dilution was used in 5% NFDM/TBST at 4 ℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:5,000 dilution was used for 2 hour at room temperature.
ET1601-4_6.jpg Fig6: ICC staining of GAPDH in A549 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-4, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1601-4_7.jpg Fig7: ICC staining of GAPDH in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-4, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1601-4_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-GAPDH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1601-4_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded mouse spleen tissue using anti-GAPDH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1601-4_10.jpg Fig10: Flow cytometric analysis of GAPDH was done on HepG2 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1601-4, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).
ET1601-4_11.jpg Fig11: Western blot analysis of GAPDH on zebrafish tissue lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/5,000 dilution.

Lysates/proteins at 40 µg/Lane.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Exposure time: 14 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1601-4_12.jpg Fig12: Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1601-4_13.jpg Fig13: Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1601-4_14.jpg Fig14: Immunohistochemical analysis of paraffin-embedded rat liver tissue with Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1601-4_15.jpg Fig15: Immunocytochemistry analysis of HeLa cells labeling GAPDH with Rabbit anti-GAPDH antibody (ET1601-4) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-GAPDH antibody (ET1601-4) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1601-4_16.jpg Fig16: Immunocytochemistry analysis of NIH/3T3 cells labeling GAPDH with Rabbit anti-GAPDH antibody (ET1601-4) at 1/2,500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-GAPDH antibody (ET1601-4) at 1/2,500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1601-4_17.jpg Fig17: Flow cytometric analysis of HeLa cells labeling GAPDH.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-4, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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