The cells were harvested and lysed with RIPA lysis buffer. Equal amounts of protein were loaded and separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred onto 0.4-µm polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk for 3 h, the PVDF membranes were incubated overnight with primary antibody against RUNX (H00000860-M06) or GAPDH (H00002597-M01) (1:1,000; Abnova, Taipei, China) diluted in blocking solution. Subsequently, the membranes were washed 3 times with Tris-buffered saline (TBS) with Tween-20 (TBST) [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween-20] and horseradish peroxidase-conjugated secondary antibodies (ZB-2305; ZSGB-BIO, Beijing, China) were incubated with the membranes for 1 h at room temperature. The membranes were washed twice with TBST and once with TBS, and soaked in enhanced chemiluminescence (ECL) reagent. Protein bands were visualized using the Western Blotting Systems ECL kit (Amersham, Piscataway, NJ, USA). Data obtained from western blot analysis were analyzed using Bio-Rad Quantity One 1-D Analysis software (Bio-Rad, Hercules, CA, USA).
Western blotting systems ecl kit
Manufactured by Cytiva
Sourced in United States
The Western Blotting Systems ECL kit is a laboratory equipment product designed for the detection and analysis of specific proteins in biological samples. The kit provides the necessary reagents and materials for performing enhanced chemiluminescence (ECL) Western blotting, a widely used technique in molecular biology and biochemistry.
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2 protocols using western blotting systems ecl kit
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Osteogenic Markers
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The experimental procedures were performed as per a previously described method.[21 (link)] Briefly, the cells were harvested and lysed with the RIPA lysis buffer. Separated proteins were transferred to 0.4-μm polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% skim milk for 3 hours, and then incubated overnight at 4°C with one of the primary antibodies (ALPL (11187-1-AP, 300 μg/ml, Proteintech, China), PDGFA (E13750, 200 μg/ml, Spring Bioscience, USA), BMP2 (18933-1-AP, 250 μg/ml, Proteintech, China), or GAPDH (H00002597-M01) (1:1,000; Abnova, Taipei, China) diluted in blocking solution. The blots were washed 3 times with Tris-buffered saline with Tween-20 (TBST) and then incubated with horseradish peroxidase-conjugated secondary antibodies (ZB-2305, 80 μg/200 μl, ZSGB-BIO, Beijing, China) for 1 hour at room temperature. Next, the blots were washed twice with TBST, once with Tris-buffered saline (TBS), and then incubated in enhanced chemiluminescence (ECL) reagent for 5 minutes. Protein bands were visualized using the Western Blotting Systems ECL kit (Amersham, Piscataway, NJ, USA). Image J software was used to qualify the scanned results.
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