我用的是条件培养基培养原代细胞，条件培养基中要添加Dexamethasone、Insulin、Glucagon、Transferrin等物质。按照条件培养基的配制要求，一般不能够应用临床上的药物，因为级别和纯度不一致，not for research only，这是配制条件培养基的最基本要求。所以你应该选用for research only的试剂，可以选用Sigma的细胞培养试剂，我用的胰岛素是I6634.作者: cj_mondy 时间: 2012-2-22 15:30
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INSULIN from Bovine Pancreas
Sigma Prod. No. I5500, I6634, I1882, and I4011
METHOD OF PREPARATION:
Insulin is purified from an ethanol/phosphoric acid extract of bovine pancreas by multiple ammonium sulfate and
isoelectric precipitations. It is then crystallized without zinc followed by crystallization with zinc. The zinc content of
the final product is approximately 0.5%. Zinc can be removed by solubilizing the insulin in dilute acetic acid, adding
excess EDTA to chelate the zinc, and then precipitating the insulin at its pI.7
STABILITY / STORAGE STABILITY:
Insulin should be stored protected from light in a desiccator at -20°C.
SOLUBILITY / SOLUTION STABILITY:
Insulin has low solubility at neutral pH. It can be solubilized at 2 mg/ml in dilute acetic or hydrochloric acid, pH 2-3.
A stock solution can be stored frozen at -20°C in single-use aliquots. Freeze-thaw cycles should be avoided.
Alternatively, it can be stored for up to 12 months at 2-8°C if it is sterile filtered through a low protein binding
membrane or if it contains a suitable bacteriostat, such as 0.1% thimerosal or sodium azide. Insulin solutions cannot
be autoclaved. Insulin can also be solubilized in 125 mM NaHCO3.6 However, alkaline stock solutions are not
recommended since high pH increases the rate of deamidation and aggregation.
GENERAL REMARKS:
Insulin is produced in vivo in the pancreatic b-cells. The precursor protein (preproinsulin) contains a 23-30 amino
acid signal peptide attached to the amino terminal of proinsulin. Proinsulin is composed of the insulin B-chain
followed by a connecting peptide (C-peptide) and the A-chain. The signal peptide assists in translocatiing
preproinsulin into the lumen of the endoplasmic reticulum, after which it is rapidly cleaved. Proinsulin is then
transported to the Golgi complex where it is packaged into granules and converted to insulin. On secretion,
equimolar amounts of insulin and C-peptide are released into the blood.8,9,10
Insulin is the primary hormone responsible for controlling the cellular uptake, utilization and storage of glucose,
amino acids, and fatty acids while inhibiting the breakdown of glycogen, protein, and fat. Several excellent reviews
of the biochemistry, physiology, and pharmacology of insulin have been published.9,10,11,12 The insulin receptor is a
tyrosine kinase that phosphorylates 185 kDa insulin receptor substrate (IRS-1) found in most cell types. IRS-1
activates phosphatidylinositol 3 kinase (IP3 Kinase).13,14,15 The Kd for insulin at its receptor is approximately 0.5 nM
(approximately 2.9 ng/ml).15
Bovine insulin is often included as a medium supplement for cell culture. The concentration range is 1-10 mg/ml
depending on the cell type.16,17作者: misswu61 时间: 2012-2-22 15:34
