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Mills Lab Protocol: Bradford Method Rev. 10.1029

Determining Protein Concentration ­ Bradford Method 1. Mix ddH2O and Bradford Reagent according to manufacturer's instructions a. For Bio-Rad reagent mix at a ratio of 4:1 water to Bradford reagent b. For Piece Pre-mixed Reagent (cat. No. 23200) do not dilute. Use reagent directly. 2. Add 2-30µl protein extract (depending on expected concentration, and amount of extract available) to Bradford assay reagent and mix by gently inverting several times. Transfer Bradford reagent/protein extract mix to spectrophotometer cuvette Measure OD595 using visible light setting on spectrophotometer or nanodrop spectrophotometer a. Start by calibrating the instrument. Usually it will ask you to "zero the instrument" using a blank (i.e. Bradford assay reagent without protein extract added) b. Measure the OD595 to confirm that the instrument is properly zeroed c. Measure samples 5. Determine protein concentration by comparing measured OD595 values against a freshly generated standard curve (see next page).

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Mills Lab Protocol: Bradford Method Rev. 10.1029

Generating a standard curve Prepare a series of BSA concentration standards ranging from 10µg/ml to 2 mg/ml using BSA standard supplied with the Bradford reagent or using freshly dissolved BSA. Do not use BSA supplied with restriction enzymes. Add BSA standard dilutions to Bradford reagent prepared above (steps 1-2) Measure standard protein concentrations as above (steps 3-4) Create a standard curve by graphing the measured OD595 as a function of the known protein concentrations Notes: it is best to create a new standard curve each time you determine protein concentrations. However, if accuracy is not critical, an older standard curve can be used, provided that the same spectrophotometer used to make the standard curve is also used to measure extract concentration * Bradford Assay results can be unreliable when extracts contain a high concentration of SDS (such as found in RIPA buffer). If the protein concentration is low, the SDS will predominate, and results will be very inaccurate. In cases where SDS is present in the extract, it may be advisable to use a different assay, such as the Lowrie method. ·

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