BE115+-+Lab06 - Lab 6: Protein Electrophoresis (SDS-PAGE) 1...

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Page 1 of 10 Bioengineering 115: Cell Biology for Engineers Laboratory 6: Protein Electrophoresis (SDS-PAGE) Last updated: January 21, 2011 Lab 6: Protein Electrophoresis (SDS-PAGE) 1 Introduction Protein electrophoresis is an extremely popular technique in molecular biology. Simply, proteins (typically from a cell or tissue lysate) in an SDS-containing buffer are added to the top of a polyacrylamide gel. The SDS, a powerful anionic surfactant, serves to surround the protein, overwhelming its inherent charge. The protein surrounded by the negatively-charged SDS has a net negative charge approximately proportional to its mass. When a potential difference is applied across the gel, the negatively charged proteins migrate through it. Smaller proteins migrate more quickly through the gel, and the proteins are separated by size into ‘bands’. Sodium Dodecyl Sulfide – PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is commonly followed by either total protein staining or transfer and Western blotting. 2 Objectives To separate proteins in a cell lysate by molecular weight To prepare a gel for Coomassie staining To prepare a gel for transfer and Western blotting 3 References and Links SDS-PAGE simulation (http://people.rit.edu/pac8612/electro/Electro_Sim.html) The SDS-PAGE Hall of Shame (http://www.ruf.rice.edu/~bioslabs/studies/sds- page/sdsgoofs.html) Early Days of Gel Electrophoresis (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1206310/) Image J
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Page 2 of 10 Bioengineering 115: Cell Biology for Engineers Laboratory 6: Protein Electrophoresis (SDS-PAGE) Last updated: January 21, 2011 4 Reagents, Supplies, and Equipment Note: Acrylamide/Bis-acrylamide is a neurotoxin! 4.1 Reagents 1. Bio-Rad (456-1023) Tris-HCl Ready Gel Precast Gel, 7.5% resolving gel, 4% stacking gel, 10-well, 30 µ l 2. 10X gel-running buffer This buffer is diluted to 1X with dH 2 O to make gel running buffer. Gel running buffer is used to fill the electrophoresis cell (or bath), keeping the gel wet and allowing the electrophoresis unit to operate. (10x)25 mM Tris base 60.6 g Tris base (10x)192 mM glycine 288 g glycine (10x)0.1%(w/v) SDS 20 g SDS pH 8.3 Add dH2O to solids until total volume is 1800 mL, bring to pH 8.3, then add more dH2O for final volume of 2000 mL. Store at room temperature. 3. 2X SDS-PAGE sample buffer (Invitrogen - LC2676) with 10% β -mercaptoethanol This is the buffer which protein samples are diluted before being loaded into the gel for analysis. It is supplemented with β -mercaptoethanol to reduce the proteins. 4. Calf serum sample, Molecular Weight standard (provided by instructor) and protein samples from Lab 5. 5. Transfer buffer - Do NOT pH, make fresh on the day of use The transfer buffer is used when transferring proteins from the gel that you poured to a nitrocellulose membrane (for Western blotting). 25 mM Tris base
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Fall '11 term at City College of San Francisco.

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BE115+-+Lab06 - Lab 6: Protein Electrophoresis (SDS-PAGE) 1...

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