Currently i am following this protocol:
1mg/ml collagen, 0.5 mg/ml collagen in 10% resolving gel.
2x15 mins; 2.5% triton X-100 wash
incubation buffer (50mM tris, pH-7.4, 10mM cacl2, 0.05% Brij 35) at 37 0C, 20 hours. I am not getting any results.
I am now interested in Matrix metalloproteinase (MMP), since this enzyme is not detected in normal hepatocyte cell (Hc).
Hepatoma HepG2 (without fucoidan; cancer state) has Metallocarboxypeptidase D at 2.4 μg/mg of cell protein and Metalloproteinase inhibitor 4/Tissue inhibitor of metalloproteinases 4 at 0.85 μg/mg of cell protein.
Cured hepatoma HepG2 (with fucoidan; normal state) has Matrix metalloproteinase-19 at 1.3 μg/mg of cell protein. This result suggests that three day’s therapy by fucoidan may not be sufficient to cure, and three weeks of therapy may be required to completely cure the cancer.
LC liver tissue (with leprosy, adult female) has RNA replicase polyprotein (Turnip yellow mosaic virus; TYMV) at 16.2 μg/mg of cell protein, and also has Metalloproteinase inhibitor 1/Tissue inhibitor of metalloproteinases 1/TIMP-1/EPA at 1.6 μg/mg of cell protein.
Serum of 7mo boy (after biotin therapy for 13w) has Ras-related nuclear protein 1 (Solanum lycopersicum; tomato) at 8.1, Putative endonuclease FLJ39025 (Cajanus cajan; pigeon pea) at 22.2, Plasma membrane fusion protein prm-1 (Neurospora crassa; fungus) at 9.0, and Vacuolar segregation protein pep7 (Schizosaccharomyces pombe; yeast) at 2.8 μg/mg of serum protein, respectively. This serum also has Matrix metalloproteinase-15 at 1.9 μg/mg of serum protein and Dedicator of cytokinesis protein 3/Modifier of cell adhesion/Presenilin-binding protein at 3.9 μg/mg of serum protein.
Opportunistic infections of plant-related microbes in these two patient may be occurred by some virus infection (which prefers female), and infected virus may have induced genes of MMPs and MMP inhibitors.
By the way, MMPs and MMP inhibitors are all hydrophobic membrane proteins, and Triton X-100 and Brij 35 seem to be too low power to dissolve hydrophobic membrane proteins. Therefore, please use Nonidet P-40 and/or Brij-58.
PAGE (polyacrylamide gel electrophoresis) is not suitable to hydrophobic proteins (please see file; IEF for hydrophobic protein).
Chandra Somasundaram; from 5% of stock solution of collagen i added 100 ul (0.1%) in total 5 ml volume of 10% resolving gel.
Kou Hayakawa; here i elute proteins from Schirmer's strip ( a kind of filter paper absorbing tears) in ddH2O.Also I use sample from human blood serum. Is it that the collagen substrate : mmp1 enzyme ratio needs to be 50:1 . or the resolving gel has to be 7.5% ( then what will be its reason behind this)
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