Leukemia Inhibitory Factor Protein, Recombinant rat

Leukemia Inhibitory Factor (LIF) is a lymphoid factor which promotes long-term maintenance of embryonic stem cells by suppressing spontaneous differentiation. LIF has a number of other activities including cholinergic neuron differentiation, control of stem cell pluripotency, bone and fat metabolism, mitogenesis of certain factor dependent cell lines and promotion of megakaryocyte production in vivo.

Product Source: rtLIF is expressed in E. coli as a fusion protein with GST using the pGEX expression system, cleaved from GST moiety with thrombin and purified by ion-exchange chromatography.

Rat LIF (rtLIF) is a 22.1 kDa protein containing 202 amino acid residues that exhibits 91% amino acid sequence identity with murine LIF(Takahama etal 1998).Analysis of the secondary structure of rat and murine LIF indicates a difference in the two dimensional structure of both proteins. Studies have shown that rtLIF is more effective in maintaining the undifferentiated phenotype of rat ES cells than similar concentrations of murine LIF (Takahama, et al 1998).

RECOMMENDED QC PROTOCOL

M1 Bioassay

1.The M1 bioassay is performed using in vitro semi-solid agar cultures, which contain approximately 100 cells in 1 mL volumes of DME containing 20 % FCS in 0.3% agar.

2.Add 100 μL of sample or rtLIF (10(e4) units/mL in 5% FCS in isotonic saline) in two-fold serial dilutions in duplicate to 35 mm petri dishes.

3.Add 100 μL of 5% FCS in isotonic saline to two control slides.

4.Incubate at 37°C in fully humidified atmosphere of 10% CO2 in air for 7 days.

5.Score the number of colonies that show differentiation (note: 50 units is defined as the amount of activity which results in 50% of the colonies being differentiated).

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Specific Activity: 50 units is defined as the amount of rat LIF required to induce differentiation in 50% of the M1 colonies in 1 mL agar cultures.

Liquid in 50mM sodium phosphate/1mM DTT / 10% glycerol / 250mM NaCl, pH 7.4 and 0.02% Tween 20. No preservativies added.

Rat LIF is shipped on dry ice. Maintain at -20°C until expiration date. Further dilutions should be made into buffer or medium to which protein (e.g., 1% BSA) or Tween 20 has been added and stored at -20°C. Freeze thawing is not recommended.

Specific Activity: The activity of rat LIF is determined by the ability to induce differentiation of M1 myeloid leukemic cells.The minimum detectable concentration of rat LIF in this assay is 0.5 ng/mL

Tested negative in both aseptic and mycoplasma tests.

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