F23 Lab 6 SDS-PAGE Protocol

During successufl purification of every enzyme, the following may be expected: 1. Solubility in NaCl increases 2. The activity increases 3. The specificity increases 4. The number of subunits increases 5. the epitope number increases

What are the advantages of gel filtration as a technique for protein purification? Identify two types of gels (sieving matrix) that may be used for gel filtration chromatography and discuss the types of samples which may be used for each. Discuss the principle behind affinity chromatography and differentiate it from gel filtration chromatography.

topic: Isoelectric Precipitation   Appearance of the mixtures containing casein mixed with different buffer solutions: (a) TestTube 1 containing Glycine-HCl buffer; (b) Test Tube 2 containing Acetate buffer; and (c) Test Tube 3containing Phosphate buffer. QUESTION: Based on the given results, what do you think is the isoelectric pH of casein? Briefly discuss your basis for determining it.

Example of a Protein Purification Scheme: Purification of the Enzyme Xanthine Dehydrogenase from a Fungus Volume Total Total Specific Percent Fraction (mL) Protein (mg) Activity Activity Recovery 1. Crude extract 2. Salt precipitate 3. Ion-exchange chromatography |4. Molecular-sieve chromatography 5. Immunoaffinity chromatography 3,800 22,800 2,460 0.108 100 165 2,800 1,190 0.425 48 65 100 720 7.2 29 40 14.5 23 1.8 275 152.108 11 Calculate the specific activity of step#4. Note that percent recovery=% Yield.

22-A. Consider a chromatography experiment in which two compo- nents with retention factors k, = 4.00 and k2 = 5.00 are injected into a column with N = 1.00 × 10° theoretical plates. The retention time for the less-retained component is t,1 = 10.0 min. (a) Calculate fm and 1,2. Find w12 (width at half-height) and w (width at the base) for each peak. (b) Using graph paper, sketch the chromatogram analogous to Figure 22-7, supposing that the two peaks have the same amplitude (height). Draw the half-widths accurately.

Nonspecific elution of affinity bonded macromolecules is used in affinity chromatography explain why?

3 Acra Alrich TOI NOF 200 250 300 350 Wavelength (nm) UV/Vis spectrum of four brands of polysorbate 80, a common detergent used in protein purification. From Wuelfin, et al., "Polysorbate 80 UV/vis spectral and chromatographic characteristics-defining boundary conditions for use of the surfactant in dissolution analysis." 3. While the binding of Coomassie blue to proteins can lead to accurate measurements of protein concentration, there are situations where the absorbance values could be misleading. For example, detergents are often used in the purification of proteins. Certain detergents mixed in with the dye could lead to incorrect determinations of protein concentration. Take a look at the spectrum of the detergent polysorbate 80, above. If you had used this detergent to purify the protein in part c above, would you expect it to interfere with your concentration calculations? Why or why not? Absorbance (AU)

Another question about tecniques that im getting confused over  Can someone explain me which one must be used and why? thank you.

Question and helpful info attached

Acetaminophen Elixir 120 mg/5 mL   During the Covid-19 epidemic, the public were advised high fever was a common side effect of infection and to ensure they had acetaminophen on hand. Because of this, there was a sudden nation-wide shortage of commercial liquid acetaminophen. The local Doctor asked if you could compound a mixture for a young child with otitis media as the Mother cannot find any commercially. The Pharmacist did some research and found the following referenced formula for you to use. The Doctor would like to give 50 mL. The child weighs 16 kg.   Formulation:   Acetaminophen 120 mg 90% ethanol 0.7 mL Propylene glycol 0.5 mL Purified water 0.5 mL Glycerol qs ad 5 mL   Dissolve acetaminophen in ethanol and propylene glycol, add most of the glycerol, add water, adjust to volume with glycerol.   Strength: 120 mg/5 mL Child dose: 15 mg/kg q4-6h. Maximum 4 doses in 24 hours. Warnings: Contains 13% alcohol EUD: As per local guidelines.   Questions:   Calculate appropriate…

Protein Agarose Gel Electrophoresis Objectives: At the end of the exercise, you should be able to1. Understand the concepts of protein structures, isoelectric point of amino acids andproteins.2. Understand the principle of separation in protein agarose gel electrophoresis.3. Demonstrate laboratory techniques used in running a protein agarose gel (preparinggels, loading samples with micropippets, and electrophoresis).4. Understand the molecular difference between normal and mutant forms of betahemoglobin in patients with sickle cell disease, and how protein agarose gelelectrophoresis can be used in disease diagnosis. Comparing Migration Rates of Hemoglobin Isolated from Normal, Heterozygousand Sickle cell anemia Individuals at pH 9.2: Part B: pH 9.2, 1X buffer Materials on the supply bench:1) gel running apparatus (lid matches the tank); one comb.2) flasks for preparing agarose gel (125 ml)3) micropippets (P20) and yellow tip boxes4) plastic waste cup, markers and microcentrifuge…

42 please do not reject

ii only

A protein required 6.8 min to travel 82 cm to the detector in a 96 cm -long capillary tube with 25.4 kV between the ends. Find the apparent electrophoretic mobility. How many femtomoles of 2.4 μM protein are injected electrokinetically into a 50 μm diameter capillary at 5.0 kV for 3.0 sec. if the sample has half of the conductivity of the background electrolyte.

3

8L.Part I

Choice and Preparation of a Buffer System1. Choosing the proper buffer solution In Protein Precipitation, two liters of 5mM buffer solution with pH 5.2 is needed in the isolation of albumin. Which among the following buffer solution is best fitted for said purpose? Justify your answer.Buffer solutions                                                               pKa  Acetate buffer                                                                  4.73Tris- (hydroxymethy) aminomethane                                 8.08Phosphate buffer                                                               7.20 2. Preparation of the chosen buffer system Calculate and measure the amounts (in grams if solid and in mL if liquid) of weak acid and conjugate base needed to be able to prepare the chosen buffer system in part A above. Express your answer in useful units (that is, prepare it from practical amounts or concentrations of starting materials).

Diagnosis of covid-19 What are some future diagnostic approaches?   word limit-250only recognised websites (who,fda etc) must be used as references. Reference from punblised scientific journal/articles should be used. [Please mention the references along with answer. Otherwise it'll be of no use in my assignment.]

The purification continues with a cation exchange step in which the positively charged cytochrome C protein is separated from negatively charged DNA and other proteins. The cation exchange eluate (volume of solution collected) had a total volume of 42.0 mL and a 1.0 mL aliquot was set aside for further analysis. The following data was obtained from the 1.0 mL aliquot to quantify the protein amount and purity: The absorbance at 410 nm of the aliquot was diluted 5-fold was 0.474 (1 cm pathlength). The absorbance at 595 nm from a 1.0 mL Bradford Assay solution that was diluted by 100-fold from the aliquot was 0.195 (1 cm pathlength). Using the information given, Calculate the total protein amount in mg from the absorbance at 595 nm. Calculate the cytochrome C amount in mg from the absorbance at 410 nm using Beer’s Law.

Biochemical tests for S.simulans : (+ or -)                              Catalase                       ___+___ DNase                         ___+___ Coagulase                   ___-___ 7.5% salt                     ___+__ Mannitol fermentation___-___ 2. Cell morphology and arrangement of S.simulans ____

8910

Nursing question MD ordered morphine SO4 gr 2/5 and the pharmacist has a stock solution of gr 1/8 per milliliter of morphine SO4. How many milliliters of the stock solution is required to fill the Rx?

Calculate the Activity of an amylase enzyme  which is diluted 1:100 times with phosphate buffer and incubated for 10 minutes at 37 degree Celsius. Given are the amount of maltose [mg] = 5.05 and the volume of enzyme used for the assay as 0.5ml.

Materials - 2021FA-CHM-1O X Bb 4427577 d-fleet02-xythos.content.blackboardcdn.com/6086c260d7e8f/4427577?X-Blackboard-Expiration=1633143600000&X-Blackboard-Sig 6 / 9 100% Exercise 1: Standard Curve for Protein Measurements: A standard curve for protein concentration is often created using known concentrations of bovine serum albumin (protein). This process is called the Bradford Assay; it is a colorimetric assay. A special reagent turns blue when it binds to amino acids present in protein. The intensity of the color is best measured with a spectrophotometer (a device for comparing two light radiations, wavelength by wavelength). In the case of the Bradford Assay the greater the absorbance, the higher the protein concentration. A series of tests were performed on some samples and spectrophotometer: following measurements were obtained using a Protein Concentration (mg/ml) Absorbance (A) 0.26 0.098 0.56 0.213 0.383 0.84 1.12 0.473 1.40 0.527 TASKS: 1. Enter the data into Excel - the…

Can you answere this question please