Download Lowry Protein Assay Protocol (from Scott Hsieh) Solution A and more Exercises Biochemistry in PDF only on Docsity! Lowry Protein Assay Protocol (from Scott Hsieh) Solution A: 4 mg/mL NaOH and 20 mg/mL Na2CO3 in water Add 2 g of NaOH and 10 g of Na2CO3 to 400 mL water while stirring until completely dissolved, then adjust volume to 500 mL. Solution B: 10 mg/mL Potassium Sodium Tartrate and 5 mg/ mL CuSO4 in water Add 100 mg Potassium Sodium Tartrate and 50 mg CuSO4 (cupric sulfate) (do not weigh on Merchant lab balances) to 8 mL of water in a plastic falcon tube (do NOT use Merchant lab glassware for solutions containing copper). Shake the mixture until solids are completely dissolved, then adjust volume to 10 mL. Mix solutions A and B and store at 4˚C this is called Lowry’s solution and should be usable for up to six months. (50:1 mix of solutions A and B) General comments Before doing the assay it is important to know whether or not your samples contain interfering species. The easiest way to do this is to prepare your samples as described in step #2 but alongside also prepare a set of tubes containing your sample plus a known amount of protein (BSA) to each sample. Should there be no interfering species your curves should run roughly parallel to each other. If there is an interfering species then the curves will not run parallel. Sample Curve to Test for Interference (No interference) y = 0.055x + 1E-16 R2 = 1 y = 0.055x + 0.2 R2 = 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 2 4 6 8 10 uL Sample added Ab s. Standard alone Protein + standard Linear (Standard alone) Linear (Protein + standard) Sample Curve to Test for Interference (Interference) y = 0.055x + 1E-16 R2 = 1 y = 0.0441x + 0.2183 R2 = 0.9992 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 2 4 6 8 10 uL Sample added A bs . Standard alone Protein + standard Linear (Standard alone) Linear (Protein + standard) Assay 1) Prepare Standards as indicated below in glass tubes (16 x 100 mm). For greater accuracy run this step in duplicate. µg protein added 0 2.5 5 7.5 10 12.5 15 17.5 20 25 30 35 2mg/mL BSA (μL) 0 1.25 2.5 3.75 5 6.25 7.5 8.75 10 12.5 15 17.5 H20 (μL) 200 198.75 197.5 196.25 195 193.75 192.5 191.25 190 187.5 185 182.5 2) Prepare samples by adding 2, 5, and 10 μL of sample into a glass tube and adjust total volume to 200μL. (The volumes given above are recommended for Chlamydomonas cell extracts (per our usual freeze-thaw protocol) but can be altered depending on expected protein concentration). It is suggested to prepare two sets of sample to determine precision. 3) To each tube add 1mL Lowry’s Solution, vortex, wait 15 min. 4) To each tube add 100μL 1.0N Folin’s Phenol reagent from Sigma (F-5292) while vortexing, wait 30 min. 5) Measure A750nm. Ideally, your absorbances should be between 0.1 and 0.5.
