One μL of serum, 15 μg of whole cell lysate, 16 μL of concentrated culture media of HepG2 or 10 μL of lipoprotein solution were heated to 70 °C for 15 minutes in SDS gel-loading buffer, and applied to SDS gel electrophoresis, thereafter proteins were transferred to nitrocellulose filters (GE Healthcare, Little Chalfont, UK). The blots were incubated with rabbit anti-ApoL1 antibody (SIGMA, MO, USA) or rabbit anti-human GAPDH antibody (Abcam, Cambridge, UK), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (IBL, Gunma, Japan). Signal intensity of ApoL1 was measured by Image J.
Rabbit anti human gapdh antibody
Manufactured by Abcam
Sourced in United States
Rabbit anti-human GAPDH antibody is a polyclonal antibody raised in rabbits against the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a widely expressed and constitutively active enzyme involved in glycolysis. This antibody can be used to detect and quantify GAPDH expression in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose filter, by GE Healthcare (1 mentions)
Rabbit anti apol1 antibody, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tables, by Roche (1 mentions)
Hybond p, by Cytiva (1 mentions)
Bradford assay, by Avantor (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Quercetin dihydrate, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit antibody, by Biosharp (1 mentions)
Mouse anti human β actin antibody, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Corning (1 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Sangon (1 mentions)
Ecl kit, by Solarbio (1 mentions)
6 protocols using rabbit anti human gapdh antibody
1
ApoL1 Protein Detection in Biological Samples
2
Western Blot Analysis of FOXP2 and MDFIC Proteins
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Proteins were extracted by standard procedures as previously described [34 (link)] in the presence of Complete Protease Inhibitor Cocktail Tables (Roche Applied Science). Control cells are SK-N-MC electroporated with the pLV-U6#H1#-C9G plasmid. Proteins were wet-transferred with TransFi (Invitrogen; Life Technologies) to polyvinyl difluoride (PVDF) membranes (Hybond-P, Amersham Biosciences) for 90 min. The protein-bound membranes were blocked with non-fat dry milk in Tris-buffered saline with Tween-20 at room-temperature and then incubated overnight at 4 °C with monoclonal mouse anti-human FOXP2 or MDFIC antibodies (1/1000 or 1/500; BD Pharmigen) or with rabbit anti-human GAPDH antibody (1/500; AbCam). After several PBS-T washes the membranes were incubated for 1 h at room temperature with secondary antibodies horseradish peroxidase (HRP)-conjugated with goat anti-mouse (1/1000) and goat anti-Rabbit (1/500; Dako, Barcelona, Spain) diluted in PBS-T. After several PBS-T washes the membranes were developed with enhanced chemiluminiscence (ECL) (GE Healthcare). The ECL signals were visualized on X-ray films. The FOXP2 and MDFIC proteins were normalized with the GAPDH internal control in the same lane.
3
Immunoblotting for PDCD4 Protein
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Whole-cell extracts were prepared using M-PER lysis buffer (Pierce). The concentration of proteins was measured by Bradford assay (Amresco), and equal protein amounts were used for SDS-PAGE. Briefly, proteins from whole-cell extracts were separated and transferred onto nitrocellulose membranes. The membranes were blocked with 1× TBS-T with 5% nonfat dry milk (Bio-Rad), followed by incubation overnight at 4°C with 1:1000-diluted primary anti-rabbit PDCD4 antibody (Cell Signaling). For protein loading controls, rabbit anti-human GAPDH antibody (Abcam) was used at 1:2500 dilution in Superblock T20 buffer (Pierce). The blot was then probed with 1:10,000-diluted goat anti-rabbit IgG secondary-HRP antibody (Pierce). The signal was detected by Super Signal West Femto ECL (Pierce).
4
Quercetin Dihydrate Treatment Evaluation
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Quercetin dihydrate (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO; Corning, USA) and further diluted with medium to reach 0, 20, 40, 50, 80, 100, 150, 160, and 200 μM before treatment. The following primary antibodies were used in the present study: rabbit anti-human Fascin antibody (1:1000; Abcam, USA), rabbit anti-human Erzin antibody (1:1000; Cell Signaling Technology, USA), mouse anti-human MMP-2 antibody (1:1000; Cell Signaling Technology, USA), rabbit anti-human MMP-9 antibody (1:1000; Cell Signaling Technology, USA), mouse anti-human β-actin antibody (1:3000; Abcam, USA), rabbit anti-human GAPDH antibody (1:3000; Abcam, USA), mouse anti-human vimentin antibody (1:400; Proteintech, USA), and mouse anti-human cytokeratin antibody (1:400; Proteintech, USA). The secondary antibodies used in this study were listed as follows: peroxidase-conjugated goat anti-rabbit antibody (1:5000; Biosharp, China) and anti-mouse antibody (1:5000; Biosharp, China).
5
Quantify ROCK1 Protein Expression
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Western blotting assay was performed to detect the protein expression level. In brief, tissues and cells were solubilized in cold RIPA lysis buffer. Proteins were separated with 10% SDS-PAGE and transferred onto PVDF membrane (Thermo Fisher), which was blocked by 5% skim milk for 1 h, and then incubated overnight at 4°C with rabbit anti-human ROCK1 antibody (1:100; Abcam, Cambridge, MA, USA) and rabbit anti-human GAPDH antibody (1:200; Abcam), and then with goat anti-rabbit secondary antibodies (1:10,000; Abcam) for 40 min. An ECL kit (Thermo Fisher) was used to visualize the protein bands.
6
CD147 Expression Analysis in Doxorubicin-Treated Cells
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Cells were seeded at the rate of 5 × 10 5 cells per well with 2 mL of media into six-well plates, treated with DOX (10 and 20 µg/mL) for 24 h, and lysed with radioimmunoprecipitation assay (RIPA) buffer (Sangon Biotech) for 30 min on ice. An equal amount (20 µg) of cellular proteins was separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST buffer (10 mM Tris-HCl, pH7.5, 150 mM NaCl and 1% Tween-20) for 2 h at room temperature with constant shaking and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-human CD147 antibody (1:1000; Abcam, Cambridge, UK) and rabbit antihuman GAPDH antibody (1:5000; Abcam). Membranes were washed with TBST three times for 30 min and incubated with horseradish peroxidase-conjugated secondary antibody (donkey anti rabbit, 1:5000; Abcam) for 2 h at room temperature with constant shaking. Finally, membranes were exposed to film after enhanced chemiluminescence with ECL kit (Solarbio, Beijing, China).
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