The proteins of the placentas were extracted, and the protein concentration was determined with the bicinchoninic acid protein quantification method. Then, 30 μg of total protein was separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA) using a semidry western blot transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were incubated with primary rabbit anti-MMP-2 monoclonal antibody (1 : 1,000 dilution), rabbit anti-VEGF monoclonal antibody (1 : 1,000 dilution) (Abcam, Cambridge, MA, USA), and anti-GAPDH (loading control, 1 : 3,000 dilution) at 4°C overnight. After washing with PBST three times, the membranes were incubated with secondary HRP-conjugated goat anti-rabbit antibody (1 : 10,000 dilution; Cell Signalling Technology, Inc.) for 2 h at room temperature. Finally, the signals were developed with a chemiluminescent ECL reagent (Millipore), and the membrane was exposed to X-ray film. The strips were analysed by scanning using Image Lab software. The ratio of the protein band density to the reference GAPDH band density was determined as the relative expression level of the target protein.
Semidry western blot transfer system
Manufactured by Bio-Rad
Sourced in United States, Germany
The Semidry Western Blot Transfer System is a compact and efficient device used for the transfer of proteins from polyacrylamide gels to membranes during Western blotting procedures. It utilizes a semi-dry transfer method to facilitate the efficient and consistent transfer of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Anti vegf rabbit monoclonal antibody, by Abcam (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ecl kit, by Roche (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
2 protocols using semidry western blot transfer system
1
Western Blot Analysis of Placental Proteins
2
Protein Expression Analysis by Western Blot
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Total cellular protein was extracted after generating the stable miR-330 expressing HCT116 and SW-480 using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) according to Santa Cruz's instructions. The vertical electrophoresis performed by SDS-PAGE method. Fifty microgram of total protein from each sample loaded into the gel (4% stacking and 10% running). After that the proteins blotted into PVDF membrane (Roche, Germany) by semi-dry western blot transfer system (Bio-Rad). The membrane blocked with tween-20 (0.5% in PBS) for 2h. Subsequently, the membrane incubated with goat monoclonal antibody against HMGA2, Snail-1, and b-actin, and mouse monoclonal anti-AKT, -P-AKT, -STAT3, -P-STAT3,-E-cadherin and -VEGFR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and mouse anti-Smad3 monoclonal antibody was bought from Abnova Corporation (Taipei, Taiwan). Then the membrane incubated with rabbit and mouse anti-goat secondary antibody conjugated with HRP. The protein bands visualized by ECL kit (Roche, Germany) via Western blot imaging instrument (Sabz.co, Iran). The proteins bond density measured using ImageJ software (NIH, Bethesda, Maryland) and normalized with beta-actin bands.
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