Bovine serum albumin (BSA) is a single polypeptide chain consisting of about 583 amino acid residues and no carbohydrates. At pH 5-7 it contains 17 intrachain disulfide bridges and 1 sulfhydryl group. Theoretical BSA relative molecular mass from amino acid sequence is 66 399. Albumin is one of the most extensively used proteins in biological research today. It acts as a powerful antioxidant in cell culture.
In order to measure and plot a standard curve of protein concentration versus absorbance at 595 nm, a series of dilutions of the BSA protein standard stock solution must be prepared. Stock solutions are used to save preparation time, conserve materials, reduce storage space, and improve the accuracy with which working lower concentration solutions are prepared. The easiest way to solve for the volume of protein stock solution required for each dilution is to use the formula C1V1 = C2V2.
Problem: Construct a calibration curve by plotting the mean absorbance for each standard concentration against the target protein concentration. The concentration of the stock solution (C1) is 100 mg/L, and the volume of the diluted sample is fixed at 2 mL (V2). Protein standards should be prepared in the same buffer as the samples to be assayed. A convenient standard curve can be made using bovine serum albumin with concentrations of 0, 10, 20, 30, 40, 50 mg/L.
== SolCalc - Laboratory Report == September 30, 2012 10:46:02 BSA y(BSA) = 30 mg/L ======================= To prepare 2 mL of a 30 mg/L solution of BSA we will need to dilute 0.6 mL of 100 mg/L BSA to a final volume of 2 mL with deionized (distilled) water. SOURCE: ------------- Name: BSA Formula: BSA Formula weight: 66399 g/mol CALCULATION: ------------- The key concept is that the amount of solute in the desired solution must be equal to the amount of solute in the source solution. Remember, the concentration is the amount of a solute divided by the volume of the solution. Before we make any calculations we have to make sure that we only use one system and one unit of measurement. DO NOT mix measurement systems and units. Desired solution: V0 = 2 mL * (1 L)/(1000 mL) = 0.002 L y0 = 30 mg/L * (1 g/L)/(1000 mg/L) = 0.03 g/L Source solutions: y1 = 100 mg/L * (1 g/L)/(1000 mg/L) = 0.1 g/L -------------------------------- First, we calculate the concentration of the desired solution c0 = y(BSA) / M(BSA) c0 = 0.03 g/L / 66399 g/mol c0 = 4.51814e-007 mol/L Then, we determine the concentration of the source (stock) solution c1 = y(BSA) / M(BSA) c1 = 0.1 g/L / 66399 g/mol c1 = 1.50605e-006 mol/L Since the total amount of solute is the same before and after dilution, the volume of stock solution needed is V1 = V0 * c0 / c1 V1 = 0.002 L * 4.51814e-007 mol/L / 1.50605e-006 mol/L V1 = 0.0006 L To convert the result into a desired unit we will use dimensional analysis again V(100 mg/L BSA) = 0.0006 L * (1000 mL)/(1 L) = 0.6 mL PROCEDURE: ------------- First of all, fill the volumetric flask about halfway with deionized water to avoid violent reactions. NEVER add water to concentrated acid. Choose a clean pipette of suitable size and transfer the liquid to the volumetric flask. When the whole solution has been drained, touch the tip of the pipette to the side of the volumetric flask to allow the last of the liquid to drain out. DO NOT blow out the remaining solution. Allow the solution to reach room temperature because a volumetric flask is only accurate at the temperature at which it has been calibrated (usually 20 °C). Very carefully fill the flask to the mark on the neck of the flask, using a dropping pipette to add the last few milliliters of liquid. Mix your solution thoroughly, by inverting the flask and shaking. NEVER hold large volumetric flasks by the neck alone - provide support at the bottom. Transfer the prepared solution to a clean, dry storage bottle and label it. NEVER store solutions in a volumetric flask. SAFETY NOTES: ------------- - When making chemical solutions, always use the appropriate safety equipment. - As a general rule, always add the more concentrated solution to the less concentrated solution. - All chemicals that you are unfamiliar with should be treated with extreme care and assumed to be highly flammable and toxic. DISCLAIMER: ------------- Use SolCalc at your own risk! If you don't understand the results, DON'T use them. ======================= https://www.periodni.com
Citing this page:
Generalic, Eni. "SolCalc Help: Diluting protein solution." EniG. Periodic Table of the Elements. KTF-Split, 27 Oct. 2022. Web. 5 Jan. 2025. <https://www.periodni.com/enig/solcalc_help/diluting_protein_solution.html>.
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