CM were collected and stored at −80°C, and cells remaining on the culture dish were counted for normalization. Protein concentrations were measured using Bradford reagents. Samples were incubated at 95°C for 10 min, loaded on 4%–15% gradient tris-glycine SDS-polyacrylamide gels (Invitrogen), separated by electrophoresis and transferred to PVDF membranes. Membranes were blocked in TBST/BSA for 1 h at room temperature, probed overnight at 4°C with primary antibodies in blocking buffer, washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using Supersignal® West chemiluminescent substrate (Thermo Scientific). Densitometry was performed using ImageJ by the measurement of area under the curve method following background subtraction.
Gradient tris glycine sds polyacrylamide gels
Manufactured by Thermo Fisher Scientific
4%–15% gradient tris-glycine SDS-polyacrylamide gels are laboratory equipment used for protein separation and analysis. They provide a gradient of polyacrylamide concentrations, enabling the separation of proteins based on their molecular weight.
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3 protocols using gradient tris glycine sds polyacrylamide gels
1
Immunoblotting Procedure for Protein Analysis
2
Immunoblotting Procedure for Protein Analysis
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CM were collected and stored at −80°C, and cells remaining on the culture dish were counted for normalization. Protein concentrations were measured using Bradford reagents. Samples were incubated at 95°C for 10 min, loaded on 4%–15% gradient tris-glycine SDS-polyacrylamide gels (Invitrogen), separated by electrophoresis and transferred to PVDF membranes. Membranes were blocked in TBST/BSA for 1 h at room temperature, probed overnight at 4°C with primary antibodies in blocking buffer, washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using Supersignal® West chemiluminescent substrate (Thermo Scientific). Densitometry was performed using ImageJ by the measurement of area under the curve method following background subtraction.
3
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer, lysates were sonicated (10 s) and clarified by centrifugation, and protein concentrations were measured using Bradford reagents. Samples were incubated at 95 °C for 10 min, loaded on 4–15% gradient tris-glycine SDS-polyacrylamide gels (Invitrogen), separated by electrophoresis and transferred to PVDF membranes. Membranes were blocked in TBST/BSA for 1 h at room temperature, probed overnight at 4 °C with primary antibodies in blocking buffer, washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using Supersignal® West chemiluminescent substrate (Thermo Scientific).
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