From this standard curve and chart below, does the separation of molecules in the mixture appear successful from the gel filtration? Is there a clearly defined separation between molecules? Explain your conclusions.   Parameters required for calculation of coefficient (Kd) for unknown protein   Volume eluted (mL) Which variable does this volume represent in the equation for Kd? Fraction with maximal DNP-Aspartate detected 36 Vt Fraction with maximal Protein detected 24 Ve Fraction with maximal Blue dextran detected 6 Vo

A protein gives a single band on SDS gel electrophoresis, as shown in lanes 1 and 2 below. There is little, if any, effect from adding β-mercaptoethanol(BME) to the sample; if anything, the protein runs a little bit slower. When treated with the proteolytic enzyme thrombin and electrophoresis in the absence of BME, the protein migrates a bit more rapidly (lane 3). But if BME is present, two much more rapidly migrating bands are found (lane 4). Explain these results in terms of a model for the protein.

8L.Part I

The Bradford reagent gives a linear response only from 0.1 mg/mL to 1.4 mg/mL of protein concentration. Would we be able to use an "absorbance vs. concentration" line graph for samples outside of the range indicated? Justify your response.

Please answer the question below and show all your work. You are given a pure protein sample to characterize and provided the following information: Its molar extinction coefficient, ε280, is 0.25 liters micromole^-1 cm^-1 Using a 0.5 cm pathlength cell, you measure the absorbance at 280 nm of a 20- fold dilution of your pure protein in solution (by this, we mean that 50 ul of the protein sample was diluted to a final volume of 1 ml) and find A280 = 0.40. What is the original concentration of the protein before dilution?

Can we purify mCherry protein (His-tag attached) with immobilised metal ions affinity chromatography (Ni-IDA)? If so, how can we confirm whether the protein is purified and how to increase its purity?

Find a method that uses some form of HPLC for the analysis of proteins. What was the stationary phase used? How does this kind of stationary phase separate the proteins? What kind of mobile phase was used?  Was the method isocratic or was a gradient used? How were the proteins detected?

"If you count 70 colonies from a urine culture obtained using 0.001ml calibrated loop, how many colony forming units (CFU) will be reported?"     70     700     7000     70000     700000

Which fractions measured from your gel filtration experiment had the most and least amount of protein detected? Which fraction likely contained the most myoglobin based off molecular weight and protein presence? Which fraction likely contained the most egg albumin based off molecular weight and protein presence? Which samples measured from your ion exchange experiment had the most protein detected? Calculate the approximate pI of your proteins using the ion exchange Table 2. Ion Exchange Samples Tested by Bradford Assay   Concentration BSA (μg/mL) Concentration BSA (mg/mL) Volume of Sample added to Bradford Reagent (μL) Absorbance at 595 nm Standard 1 250 0.25 50 0.32 Standard 2 125 0.125 50 0.16 Standard 3 62.5 0.0625 50 0.08 Standard 4 31.25 0.03125 50 0.04 Standard 5 15.625 0.015625 50 0.02 Blank 0.00 0.00   0.00   Table 3. Gel Filtration Fractions Tested by Bradford Assay Fraction #:…

Use correct sig figs The concentration of a purified monoclonal antibody was measured using UV280 nm. The sample was diluted (200 μL of purified antibody in 800 μL buffer) prior to analysis using a spectrophotometer. Calculate the concentration of antibody in the purified fraction if the Abs=0.95 of the diluted antibody. The molar absorptivity is known to be 191,411.6 M-1cm-1 and the molecular weight is 150 kDa. The pathlength for the cuvette is 1 cm.