Determination of protein concentration day 3 rates are changes in a quantity per unit time, as for example, the rate of an enzyme reaction expressed in units of umoles"min. spec pressed in units of umoles min-1. specific activities are rates per unit mass. in enzyme ne units of specific activity can be umoles min-mg so how do you measure the amount of an enzyme, or for that matter, any protein? carbohydrates e the protein solution to exchange all the nonvolatile salts with volatile salts, rapid- eeze and lyophilize the protein to remove water and volatile salts, weigh the protein, dissolve the protein in water, measure the absorbance spectrum and calculate an absorptivity. " ind calculate an absorptivity. this is a lot of work, justified perhaps if you produce the protein for a medicinal purpose. two-absorbance methods provide an alternative partially correcting for nucleic acid contamination and variations the types of amino acids in the protein two-absorbance measurements employ wavelengins 260 and 280 nm (warburg-christian). 235 and 280 nm (whitaker and granum, 1980) and 280 (scopes, 1974). colorimetric assays (for example, biuret, folin another alternative: however, these are susceptible to artifacts due to contamination and ys (for example, biuret, folin-lowry and bradford) are variation in amino acids amino acids. no method is perfect. systematic errors of 15% in the determination of protein weight are not uncommon. here we compare two-wavelength methods and u tml colorimetric method of bradford using a one- and two-wavelength protoeel d recombinant proteins mala the flecoat materials and equipment: bradford reagent concentrate (diluted 1: 4 with water) bovine serum albumin solution - bsa (1 mg/ml in 0.1% nacl) lysozyme solution (1 mg/ml in 0.1% nacl) 0.1% sodium chloride (w/v) - nacl test tubes cary 60 methyl acrylate cuvettes 1.5 ml-part 1.4a quartz cuvettes (not disposable) -part 1.4b cuvette washer tatooing! looong mbl linal interfering substances: 1 % (v/v) 2-mercaptoethanol - toxic 60% (w/v) saturated ammonium sulfate - corrosive 100 mm tris-hcl buffer (ph 7) 0.5 mg/ml yeast rna 2% (w/v) sodium dodecylsulfate (sds) 1 % (w/v) solution of d-glucose ming safety disposal wear safety glasses and nitrile gloves. down the drain until the corn turns blue. 1.4a bradford assay the bradford assay uses a standard curve, and hence a requirement for standard solutions. we have seen this before, generating standard curves by two methods in part 1.1. the range of protein concentrations should be 10-fold, and five solutions should suffice. the highest concentration standard is 1000 pg/ml. serial dilution using a factor of one-half produces concentrations 1000, 500, 250, 125 and 62.5 ug/ml. a serial dilution has a shortcoming in that concentrations are not evenly spaced. a more uniform spacing (1000, 750, 500, 250 and 100 ug/ml) might be better, and is easier to plot. the protocol here employs 20 l of the standard combined with 1 ml of the diluted bradford reagent, so the protein concentrations assayed are approximately 20, 15, 10, 5 and 2 pg/ml. дор\ x 100029 /mu x 2000 - 750 xv al