Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5). The incubation was stopped by adding with a volume of 4× Laemmli sample buffer, and the sample was separated on a 12% SDS-PAGE and analyzed by Western blot analysis using either anti–galectin-3 polyclonal antibody (rabbit anti-mouse antibody; 14979-1-AP; Proteintech) or anti–galectin-3 monoclonal antibody (mouse anti-mouse antibody; ab2785; Abcam).
Protocol detail
Galectin-3 Proteolytic Cleavage by MMP9
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Acetate
Anti antibody
Galectin 3
Human galectin 3
Laemmli buffer
Mmp9
Monoclonal antibody
Mouse
Nacl
Rabbit
Recombinant protein
Sds page
Tris buffer
Western blot analysis
Corresponding Organization : Wuhan University
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Variable analysis
independent variables
- Incubation of recombinant human GALECTIN-3 protein with activated recombinant MMP9
- Addition of 5 mM p-aminophenylmercuric acetate
- Analysis of the incubated sample by Western blot using anti-galectin-3 polyclonal antibody
- Analysis of the incubated sample by Western blot using anti-galectin-3 monoclonal antibody
- Incubation temperature of 37°C
- Incubation time of 30 minutes
- Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5)
- Positive control: Not mentioned
- Negative control: Not mentioned
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