Total protein was lysed in PIPA lysis buffer (Thermo Scientific) on ice for 30 minutes. The lysates were centrifuged at 12,000 rpm for 30 minutes at 4°C. Protein concentration was determined by using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (20 μg) were separated in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Then the membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for one hour and incubated with primary antibodies (SIP, 1: 1,000, ab167084; p65, 1: 1,000, ab16502; p-p65,1: 1,000, ab6503; GAPDH, 1: 1,000, ab9485; Abcam, USA) at 4°C overnight. Afterwards, the membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for one hour. The bands were detected by an enhanced chemiluminescence (ECL) kit (Beyotime, Shanghai, China). Grey levels of the bands were quantified with Quantity One software (Bio-Rad).
Ab6503
Manufactured by Abcam
Sourced in United States
Ab6503 is a polyclonal antibody that recognizes the human Laminin B1 protein. Laminin B1 is a component of the extracellular matrix and plays a role in cell adhesion and differentiation. The antibody is suitable for use in applications such as Western blot and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab136668, by Abcam (1 mentions)
Ab118973, by Abcam (1 mentions)
Ab109330, by Abcam (1 mentions)
Ab3778, by Abcam (1 mentions)
Ab219620, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
2 protocols using ab6503
1
Western Blot Analysis of Protein Expression
2
Quantitative Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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RIPA (Beyotime, Shanghai, China) was applied to isolate total protein. BCA (Thermo Fisher Scientific) was used to quantify the total protein. SDS-PAGE (10%) was used to separate proteins (40 μg per lane), and then proteins were transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with primary antibodies against aggrecan (ABCAm; ab3778, 1:1,000), MMP-1 (ABCAm; ab118973, 1:1,000), MMP13 (ABCAm; ab219620, 1:1,000), PRDX3 (ABCAm; ab136668, 1:1,000), Cyclin D1 (ABCAm; ab16502, 1:1,000), STAT3 (ABCAm; ab109330, 1:1,000), p-STAT3 (ABCAm; ab6503, 1:1,000), and β-actin (ABCAm; ab179467, 1:1,000) after blocked with 5% skimmed milk for 1 h. After that, the membranes were incubated with secondary antibodies (HRP-conjugated, ABCAm; 1:5,000) for 1 h at room temperature. Protein bands were visualized using the enhanced chemiluminescent (ECL) kit (Thermo Fisher Scientific). β-actin was used for normalization. Image-Pro Plus 6.0 was applied for densitometric analysis.
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