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BiochemicalsProteins and Peptides
Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
Low endotoxin, Azide free.
Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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In this procedure, active gelatinases digest gelatin embedded in a polyacrylamide gel. After Coomassie staining, areas of degradation are visible as clear bands against a darkly stained background.
This protocol is optimized for detecting secreted MMP-9 and MMP-2 activity in conditioned media.
Plate cells cultured in fetal bovine serum (FBS).
At 70–80% confluency, remove FBS media.
The duration of growth in FBS-free media must be optimized for the cell line: for example, 231G and 468 breast cancer cells require a 40–44 h growth period before collecting conditioned media.
Collect conditioned media and centrifuge or filter to eliminate dead cells.
Concentrate conditioned media 10X.
Recipe to prepare 250 mL of 5X non-reducing sample buffer:
Reagent | Weight/volume for 250 mL | Final concentration |
SDS | 10 g | 4% |
Glycerol | 50 mL of 100% glycerol | 20% |
Bromophenol blue | 0.025 g | 0.01% |
Tris-HCl | 4.91 g | 125 mM |
H2O | 200 mL | - |
Dilute conditioned media so that all samples have the same protein concentration.
Add 5X non-reducing sample buffer to your samples.
Prepare a 7.5% acrylamide gel containing gelatin.
Load sample to each well.
Include a protein molecular weight marker in one well.
Run the gel at 150 V until good band separation is achieved.
Final concentration | For 250 mL |
---|---|
2.5% Triton X-100 | 6.25 mL of 100% |
50 mM Tris-HCl | 12.5 mL of 1 M stock |
5 mM CaCl2 | 625 μL of 2 M stock |
1 μM ZnCl2 | 2.5 μL of 0.1 M stock |
H2O | 228 mL + 2 mL 2% NaN3 |
Final concentration | For 250 mL |
---|---|
1% Triton X-100 | 2.5 mL of 100% |
50 mM Tris-HCl | 12.5 mL of 1 M stock |
5 mM CaCl2 | 625 μL of 2 M stock |
1 μM ZnCl2 | 2.5 μL of 0.1 M stock |
H2O | 233 mL + 2 mL 2% NaN3 |
Wash the gel 2 x 30 min with the washing buffer at room temperature with agitation.
Soaking the gel in the washing buffer removes SDS from the gel.
Rinse for 5–10 min in incubation buffer at 37°C with agitation.
Replace with fresh incubation buffer and incubate for 24 h at 37°C.
The incubation buffer contains cofactors necessary for the gelatinase reaction to occur.
Stain the gel with staining solution for 30 min to 1 h at room temperature with agitation.
Rinse with water until excess staining solution is removed.
Incubate with destaining solution until bands can clearly be seen.