Journal: Proteome Science

Article Title: A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

doi: 10.1186/1477-5956-7-28

Figure Lengend Snippet: Batch Ion-Exchange of soluble C2C12 proteins increases the total number of spots visualized 2.5-fold . Serum-starved C2C12 cells were stimulated for 2 hours with 100 nM IGF-1 and lysed in detergent-free buffer to generate soluble proteins. 100 ug of protein was separated by 2D electrophoresis followed by silver staining (left). Another 100 ug of protein was further fractionated by batch ion-exchange followed by desalting/concentration before 2D electrophoresis and silver staining. The total amount of protein detected was analyzed using PDQuest software (Bio-Rad).

Article Snippet: Samples containing 75–200 μg of fractionated cytoplasmic proteins were diluted in 2D electrophoresis buffer (2 M thiourea, 5 M urea, 0.25% CHAPS, 0.25% Tween-20, 0.25% SB-3, 10% isopropanol, and 12.5% water-saturated butanol) containing 15 mg DTT and 0.5% ampholytes (pI ranges 3–10 or 5–8; Bio-Rad; Hercules, CA) to a final volume of 0.2 ml, and passively absorbed onto immobilized pH gradient strips (pI ranges 5–8 or 3–10 NL (non-linear); Bio-Rad) for 4 hours at 20°C followed by active rehydration (50 V at 20°C) for 8 hours.

Techniques: Two-Dimensional Gel Electrophoresis, Silver Staining, Concentration Assay, Software

Journal: Proteome Science

Article Title: A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

doi: 10.1186/1477-5956-7-28

Figure Lengend Snippet: Expression of Rho-GDI, Cofilin, PDGF receptor α, and MAPKBP-1 decreases upon IGF-1 stimulation of C2C12 cells . A) Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (Rho-GDI, Cofilin, PDGF receptor α, and MAPKBP-1) decrease in expression after IGF-1 treatment. PDQuest analysis indicates a quantitative change in the protein based on total quantitation of the entire gel. B) Western blot analysis confirms the sustained decrease in protein expression for Rho-GDI, cofilin, and the PDGF receptor.

Article Snippet: Samples containing 75–200 μg of fractionated cytoplasmic proteins were diluted in 2D electrophoresis buffer (2 M thiourea, 5 M urea, 0.25% CHAPS, 0.25% Tween-20, 0.25% SB-3, 10% isopropanol, and 12.5% water-saturated butanol) containing 15 mg DTT and 0.5% ampholytes (pI ranges 3–10 or 5–8; Bio-Rad; Hercules, CA) to a final volume of 0.2 ml, and passively absorbed onto immobilized pH gradient strips (pI ranges 5–8 or 3–10 NL (non-linear); Bio-Rad) for 4 hours at 20°C followed by active rehydration (50 V at 20°C) for 8 hours.

Techniques: Expressing, Two-Dimensional Gel Electrophoresis, Silver Staining, Quantitation Assay, Western Blot

Journal: Proteome Science

Article Title: A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

doi: 10.1186/1477-5956-7-28

Figure Lengend Snippet: Expression of Rad50, enolase, IκB kinase b, and Hsp70 increases upon IGF-1 stimulation of C2C12 cells . A) Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (Rad50, enolase, IκB kinase b, and Hsp70) increase in expression after IGF-1 treatment. PDQuest analysis indicates a quantitative change in the protein based on total quantitation of the entire gel. B) Western blot analysis confirms the sustained increase in protein expression for Rad50, enolase, IκB kinase b, and Hsp70. C) RAD50 accumulates in the cytosolic fraction of IGF-1 stimulated C2C12 cells.

Article Snippet: Samples containing 75–200 μg of fractionated cytoplasmic proteins were diluted in 2D electrophoresis buffer (2 M thiourea, 5 M urea, 0.25% CHAPS, 0.25% Tween-20, 0.25% SB-3, 10% isopropanol, and 12.5% water-saturated butanol) containing 15 mg DTT and 0.5% ampholytes (pI ranges 3–10 or 5–8; Bio-Rad; Hercules, CA) to a final volume of 0.2 ml, and passively absorbed onto immobilized pH gradient strips (pI ranges 5–8 or 3–10 NL (non-linear); Bio-Rad) for 4 hours at 20°C followed by active rehydration (50 V at 20°C) for 8 hours.

Techniques: Expressing, Two-Dimensional Gel Electrophoresis, Silver Staining, Quantitation Assay, Western Blot

Journal: Proteome Science

Article Title: A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

doi: 10.1186/1477-5956-7-28

Figure Lengend Snippet: Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain . The protein highlighted by a yellow square (eIF-4H) rapidly increases in expression after 1 hour IGF-1 treatment, but then decreases at 4 and 8 hours.

Article Snippet: Samples containing 75–200 μg of fractionated cytoplasmic proteins were diluted in 2D electrophoresis buffer (2 M thiourea, 5 M urea, 0.25% CHAPS, 0.25% Tween-20, 0.25% SB-3, 10% isopropanol, and 12.5% water-saturated butanol) containing 15 mg DTT and 0.5% ampholytes (pI ranges 3–10 or 5–8; Bio-Rad; Hercules, CA) to a final volume of 0.2 ml, and passively absorbed onto immobilized pH gradient strips (pI ranges 5–8 or 3–10 NL (non-linear); Bio-Rad) for 4 hours at 20°C followed by active rehydration (50 V at 20°C) for 8 hours.

Techniques: Two-Dimensional Gel Electrophoresis, Silver Staining, Expressing