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13 Dec 2019

Hiroshi is planning on running some protein assays in which he will be varying the protein amount. The total volume of protein sample that will be added to the assay is exactly 5uL. Hiroshi stock protein concentration is 2mg/mL and the amounts Hiroshi is planning to use in the assay are: 10 ug,8 ug, 4,ug, 2 ug , 1 ug and 0.5 ug. Help out Hiroshi by showing him how to make up a serial dilution of his protein stock solution so that he will be able to easily perform this assay.

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Trinidad Tremblay
Trinidad TremblayLv2
17 Dec 2019
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Related questions

You need to validate the concentration of a 50 μL stock solution reported it be 25 mg/mL based single measurement. To conserve sample you decide to use the UV-vis plate reader to make an absorbance measurement using a 100 μL well-volume.

Given:

The protein has a molecular weight of 6,343 g/mol. and a molar extinction coefficient or 113,268 M-1cm-1 (Effective pathlength = 0.3 cm for a 100 μL well volume). The instrument you have available provides accurate absorbance readings for sample absorbance in the range 0.1 - 1. 0. You have P20 and P200 pipettes and 20 mM phosphate buffer available.

a) What fold dilution of the sample would be needed get and absorbance of 0.5?

b) Describe how you dilute 2 μL of the 25 mg/mL sample so that this absorbance reading can be made.

c) After completing a single measurement of the sample your calculations indicate the sample is 26.7 mg/mL. What percent agreement exists between your determination and the previously reported value? What factors are most likely?

d) You need to assay the purity of the sample from parts A-C by SDS-PAGE. Explain how to prepare a 20 μL sample containing 30 μg for this experiment.

After combining with 2x sample loading buffer and heating, you transfer 25 μL of the denatured protein into a well of the resolving gel.

Compute the total amount of protein that was added to the gel in micrograms and nanomoles.

HELP WITH PART D) E)

Question: STANDARD CURVE PREPARATION Always indicate the volume of water (or additional solution) to be add... STANDARD CURVE PREPARATION Always indicate the volume of water (or additional solution) to be added to complete any dilution.

a) A stock solution of 6mg/ml BSA is provided. Calculate the dilutions for the preparation of the following BSA standards (2.0ml volumes of each): 60, 170, 500, 1500, 3000ug/ml. Also include the dilution factor for each preparation. On a separate sheet of paper with a heading of Calculations, include one complete sample calculation of this dilution calculation.

b) To 0.5ml of your unknown samples (and dilutions of your samples) and to 0.5ml of the standards add 2.5ml of Biuret reagent. Calculate the final concentration of BSA after addition of the Biuret reagent. iv. neatly show one sample calculations.

c) Hypothetical absorbencies with respect to the concentrations in ‘a’ are: 0.16, 0.20, 0.28, 0.45 and 0.70. v. Use computer software such as excel to plot a standard curve using these absorbencies against the concentration of each BSA standard prior to the addition of Biuret reagent (use concentrations as given in step 11a directly) vi. Label the figure as Figure 1 and give it an appropriate descriptive title. The title should be above the figure. vii. Plot a second standard curve using the same absorbencies (0.16, 0.20, 0.28, 0.45 and 0.70) against the concentration of the BSA standard after the addition of the Biuret reagent (as calculated in step 11b) viii. Label this figure as Figure 2 and give it an appropriate descriptive title.

d) A sample prepared with 0.50ml of protein sample A and 2.5ml of Biuret reagent gave an absorbance of 0.51. Calculate the protein concentration of the original protein A sample, in g/ml, twice: once with each standard curve. ix. On the calculation sheet (see iii), neatly show all the calculations required to solve for the concentration of the protein sample. Remember to perform this calculation twice: once with each standard curve. Include a title to separate this calculation from others on the page.

e) A protein sample was prepared with 5.0 x 10-2 ml protein sample B, 0.45ml water and 2.5ml of Biuret reagent . It had an absorbance of 0.22 in the spectrophotometer. With this data, calculate the protein concentration of the original protein B sample, in g/ml, using either standard curve. x. On the calculation sheet (see iii), neatly show all the calculations required to solve for the concentration of the protein sample. Include a title to separate this calculation from others on the page. Refer to the Figure that was used to solve for the concentration.

The standard curve equations are:

y=0.0002x+0.1706

y=0.0011x+0.1706

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