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Anti gapdh

Manufactured by Abcepta
Sourced in United States

Anti-GAPDH is a laboratory reagent used to detect the presence and abundance of the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein in biological samples. GAPDH is a commonly used housekeeping gene and its expression is often analyzed as a reference point in various research applications.

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11 protocols using anti gapdh

1

Quantification of VEGF Expression in Engineered Cells

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H9C2 and C2C12 cells were seeded in 10-cm plates at a density of 4×105cells/well and cultured with serum-free DMEM for 24 h. At 70% confluence, Lenti-MLC-2v-VEGF165-IRES-NIS and Lenti-EF1a-VEGF165-IRES-NIS were added (MOI = 40).
Lysates of lentivirus-infected cells were prepared by standard methods. Western blot analysis was then performed by incubating the filter with mouse anti-NIS (1:500; Neomarker), anti-His-tag (1:500; Neomarker) and anti-GAPDH (1:10000; Abgent) antibody in Tris-buffered saline Tween-20 overnight at 4°C, followed by incubation with peroxidase-conjugated goat anti-mouse immunoglobulin G (1:2500; Neomarker) for 1 h at room temperature. Immunodetection was carried out using an ECL Western blot detection kit (Pierce).
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2

Phospho-protein Detection Protocol

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The indicated cells were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitor Cocktail (Sigma, Mo, USA). Supernatants were collected and subjected to 10% SDS-PAGE, and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were then incubated overnight with primary antibodies. Anti-phospho-AktSer473, anti-phospho-Erk1/2, anti-total Akt and anti-total Erk were purchased from Bioworld Technology, co, Ltd. Anti-EHF was purchased from Abcam Biotechnology, Inc. Anti-GAPDH was purchased from Abgent, Inc. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and antigen-antibody complexes were visualized using the Western Bright ECL detection system (Advansta, CA).
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3

Western Blot Analysis of β-Catenin, HIPK2, and LAMINB1

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Western blotting was completed as documented previously (13 (link)). Harvesting of proteins was performed as illustrated in the figures, fractionated via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membranes. The membranes were blocked in 1% Bovine Serum Albumin (BSA) for 20 minutes at room temperature (RT) then inoculated overnight with anti-β-catenin, anti-HIPK2, anti-GAPDH (Abgent, USA), and anti-LaminB1 antibodies. Afterwards, the blots were probed for 1 hour at room temperature with horseradish peroxidase-linked secondary antibodies before being processed with the enhanced chemiluminescence plus Western Blotting Detection System. GAPDH was utilized as a loading control.
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4

Protein Extraction and Western Blot Analysis

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Protein was extracted using a protein extraction kit (KeyGen Biotech, KGP250-2100, Nanjing, China) according to the manufacturer's instructions. The primary antibodies used were anti-RASSF8 (Sigma, HPA038163), anti–VEGF-C (R&D, AF752), anti–phospho-AKT (Cell Signaling, Danvers, MA, USA, 4691S), anti-AKT (Cell Signaling, 4060S), anti–phospho-ERK1/2 (Cell Signaling, 4370P), anti-ERK (Cell Signaling, 9102S), anti-p65 (Cell Signaling, 6956S), anti–β-tubulin (Cell Signaling, 2128S), and anti-GAPDH (Abgent, San Diego, CA, USA). Anti–lamin B1 (KeyGen Biotech, KGAA004-2) was used as the control nuclear protein. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) and a Tanon 5200 Luminescent Imaging Workstation (Tanon, Shanghai, China).
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5

Western Blot of Baculovirus-Expressed Proteins

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Lysates of baculovirus-infected cells were prepared by standard methods. Western blot analysis was then performed by incubating the filter with mouse anti-NIS (1∶500; Millipore), anti-His-tag (1∶500; Abgent) or anti-GAPDH (1∶10000; Abgent) antibody in Tris-buffered saline Tween-20 overnight at 4°C, followed by incubation with peroxidase-conjugated goat anti-mouse immunoglobulin G (1∶2500; Santa Cruz Biotechnology) for 1 h at room temperature. Immunodetection was carried out using an ECL Western blot detection kit (Pierce).
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6

Immunoblotting Analysis of Signaling Pathways

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Whole cell lysates were prepared for immunoblotting as described previously.17, 18, 19 Equal amounts of total proteins (30 μg) were subjected to SDS‐PAGE separation, followed by immunoblotting analysis with specific antibodies. Primary antibodies against p‐p65 (Ser536), p65, p‐IκBα (Ser32), IκBα, p‐IKKα/β (Ser176/180), IKKα, IKKβ, PARP, Cleaved‐Caspase 3, Bcl‐2, MCL1, XIAP and Bim were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐GAPDH was purchased from Abgent (Suzhou, China). Anti‐mouse IgG and anti‐rabbit IgG HRP conjugated antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
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7

Western Blot Analysis of Protein Signaling

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WB analysis was performed as previously described [18 (link)]. First, the lysed cell supernatant was run on SDS-PAGE and then blotted with antibodies. Antibodies used in WB were as follows: anti-GP73 (Abcam, Cambridge, UK), anti-P65 (Cell Signaling Technology, Boston, MA, USA), anti-phosphorylated P65 (ImmunoWay Biotechnology Company, Plano, TX, USA), anti-IκBα, anti-phosphorylated IκBα, anti-IKKα/β (Santa Cruz Biotechnology, CA, USA), anti-GAPDH, and anti-β-actin (Abgent, San Diego, CA, USA).
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8

Protein Expression Analysis by Western Blot

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Cells were lysed in prechilled RIPA buffer containing protease inhibitors. Equal amounts of protein lysates were separated by SDS–PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were then incubated with primary antibodies. Anti-AIB1 and anti-total-Erk1/2 (t-Erk) were purchased from Abcam, Inc. Anti-phospho-AktSer473, anti-phospho-Erk1/2 and anti-total-Akt (t-Akt) were purchased from Bioworld Technology, co, Ltd. Anti-total-Rb (t-Rb) and anti-phospho-RbS811 (p-Rb) were purchased from Epitomics, Inc. Anti-GAPDH was purchased from Abgent, Inc. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA).
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9

Western Blot Analyses of Signaling Proteins

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Cells were lysed in prechilled RIPA buffer containing protease inhibitors. The protein lysates were separated on SDS–PAGE and then transferred to PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc). The membranes were then incubated with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA).
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10

Immunoblotting Analysis of Cellular Signaling

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The indicated cells were lysed in RIPA buffer containing protease inhibitors. Supernatants were collected and subjected to 10% SDS-PAGE, and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were then incubated overnight with primary antibodies. Anti-N-cadherin was purchased from Abcam Biotechnology, Inc. Anti-phospho-Erk1/2, anti-phospho-AktSer473, anti-total Erk and anti-total Akt were purchased from Bioworld Technology, co, Ltd. Anti-p16, anti-phospho-Rb and anti-Rb antibodies were purchased from Abcam. Anti-GAPDH was purchased from Abgent, Inc. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and antigen-antibody complexes were visualized using the Western Bright ECL detection system (Advansta, CA).
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