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Protein Purification

Determining Protein Concentration

SDS Gel Electrophoresis

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DETERMINING PROTEIN CONCENTRATION
 

Procedures:
Materials Used

§         Protein Assay Dye Reagent Concentrate (catalog number 500-0006), contains 450ml of solution containing dye, phosphoric acid and methanol      

§         Bovine serum albumin (BSA), (KitII, catalog number 500-0002)
 

§         Spectrophotometer set to 595nm        

§         Cuvettes with 1 cm path length matched to laboratory spectrophotometer

§         13x100 mm test tubes

§         Test tube rack for 13x100 mm test tubes

§         Graduated cylinders, pipets, and containers for reagent preparation and storage

§         Pipets accurately delivering 100μl and 5.0 μl
 

• 1) Reconstituting the protein standard
  • Add 20 ml of deionized water to bovine serum abumin standard (BSA)
  • Mix until dissolved
  • If substance is not used within 60 days, aliquot sample and freeze at -20˚C

• 2) Prepare dye reagent
  • Dilute 40 ml of Dye Reagent Concentrate with 160 ml of distilled H₂0, (1 part Dye Reagent Concentrate with 4 parts distilled, deionized water). Good for 2 weeks if kept at room temperature (25˚C)

• 3) Prepare dilutions of protein standard
  • One staff group label 5 tubes as Set1₁-0.2, Set1₁-0.4, Set1₁-0.6, Set1₁-0.8 and Set1₁-0.9
    • In tube labeled Set1₁-0.2, mix 0.145 ml of BSA and 0.855 ml H₂0, (volumes are additive since we’re basically adding H₂0 to H₂0)
    • In tube labeled Set1₁-0.4, mix 0.292 ml of BSA and 0.708 ml H₂0
    • In tube labeled Set1₁-0.6, mix 0.438 ml of BSA and 0.562 ml H₂0
    • In tube labeled Set1₁-0.8, mix 0.584 ml of BSA and 0.416 ml H₂0
    • In tube labeled Set1₁-0.9, mix 0.657 ml of BSA and 0.343 ml H₂0
  • Pipet 100 μl of each standard into a clean, dry test tube of another set labeled Set1₂-0.2, Set1₂-0.4, Set1₂-0.6, Set1₂-0.8 and Set1₂-0.9
  • Add 5 ml of diluted Dye Reagent Concentrate to tubes in Set1₂
  • Incubate tubes at room temperature (25˚C) for at least 5 minutes, not more than an hour
• 4) Determining standard curve
  • Blank spectrophotometer set at 595 nm with sample of 5ml of diluted Dye Reagent Concentrate and 100μl of H₂0
  • Measure absorbance of samples in each tube with spectrophotometer set at 595 nm and record

• 5) Determining average standard curve
  • Repeat steps #3-4 with next staff groups for new set of test tubes labeled Set 2.

• Plot concentration vs absorption values for Set 1 and Set 2 using Microsoft Excel and determine equation of linear trendline

• 6) Determining protein concentration

• Blank spectrophotometer at 595 nm with sample of 5ml of diluted Dye Reagent Concentration and 100μl of H₂0 from step #4
• Measure absorbance of 15μl TE buffer sample and 85μl H₂0 and 5 ml of diluted Dye Reagent Concentration and record
• Measure absorbance of 15μl Wash buffer sample and 85μl H₂0 and 5 ml of diluted Dye Reagent Concentration and record
• Plot both absorbances on standard curve using determined equation to determine protein concentration