Lab Quizzes

In the affinity chromatography lab which type of interaction will be utilized to purify Lactate Dehydrogenase? a. antibody-antigen b. metal ions-histidine containing proteins c. receptor-ligand d. enzyme-substrate

b. metal ions-histidine containing proteins

What is a 2 fold dilution?

1/2 stock strength= 1/2 stock +1/2 diluent

What is a 4 fold dilution?

1/4 stock strength=1/4 stock+3/4 diluent

In the Buffers and pH lab, you will be using two techniques to identify an unknown amino acid: thin layer chromatography and titration True False

True

SDS-PAGE separates proteins based on size. True False

True

Sample buffer must be added to your samples before loading them onto the gel. Sample buffer contains beta-mercaptoethanol which has a strong odor and must be used in the hood. True False

True

In gel filtration chromatography, what dye will be used to indicate the total volume of the column (Vt)? cytochrome c Vitamin B12 (cyanobalamine) phenol red blue dextran

Vitamin B12 (cyanobalamine)

How many pKa values would you predict for the amino acid aspartic acid? a. 4 b. 3 c. 2 d. 1

b. 3

For the kinetics lab, you will be determining the kinetic parameters for alkaline phosphatase from both Lineweaver-Burk and Michaelis-Menten plots. Which of the following TWO statements below are CORRECT? a. All graphs should be generated by hand plotting using the graph paper in the back of the lab manual. b. Excel must be used to create all graphs. c. The Lineweaver-Burk plot is preferred over the Michaelis plot (generated by using non-linear regression analysis) to determine kinetic parameters. d. The Michaelis-Menten plot will be generated using non-linear regression analysis using Excel. This plot is preferred over the Lineweaver-Burk plot to determine kinetic parameters because the Lineweaver-Burk plot introduces error when the data is linearized.

b. Excel must be used to create all graphs. d. The Michaelis-Menten plot will be generated using non-linear regression analysis using Excel. This plot is preferred over the Lineweaver-Burk plot to determine kinetic parameters because the Lineweaver-Burk plot introduces error when the data is linearized.

Lactate dehydrogenase is an enzyme which catalyzes an irreversible oxidation-reduction converting pyruvate to lactate. a. True b. False

b. False

Primary antibodies are not protein specific. The same primary antibody will be used to detect cytochrome c, ovalbumin, and beta-galactosidase. a. True b. False

b. False

For the Separation of Protein series of labs, what two methods will be used to determine the concentration of ovalbumin and cytochrome c in your peak fractions (select two answers!)? a. SDS-PAGE b. Pierce 660nm protein assay c. Western Blot d. Absorbance at 280nm will be measured and used to calculate the protein concentration

b. Pierce 660nm protein assay d. Absorbance at 280nm will be measured and used to calculate the protein concentration

In the Separation of Proteins Part III lab, you will be performing the technique of Western Blot. Which statement below is NOT correct? a. Primary antibodies bind to specific proteins b. The same primary antibody will be used for the cytochrome c, ovalbumin, and beta-galactosidase blots c. The secondary antibody binds to the primary antibody d. The secondary antibodies have an enzyme covalently attached to allow colorimetric detection of the protein of interest

b. The same primary antibody will be used for the cytochrome c, ovalbumin, and beta-galactosidase blots

Which of following statements below is correct? a. absorbance values have the units of cm b. absorbance values have no units c. absorbance values have the units of moles/liter d. absorbance values have the units of M/cm

b. absorbance values have no units

The tagged lactate dehydrogenase will be eluted from the column by.................. a. increasing the pH b. increasing the concentration of imidazole c. increasing the concentration of sodium ions d. increasing the concentration of nickel ions

b. increasing the concentration of imidazole

Ion exchange chromatography will be used to separate a mixture of amino acids. The amino acids will be eluted from the column using three buffers: pH 3.5 buffer, pH 6.5 buffer, and pH 10.5 buffer. The buffers must be applied to the column in a specific order. Please select the correct order below: a. pH 3.5 buffer first, pH 10.5 buffer second, and pH 6.5 buffer last b. pH 3.5 buffer first, pH 6.5 buffer second, and pH 10.5 buffer last c. pH 10.5 buffer first , pH 6.5 buffer second, and pH 3.5 buffer last

b. pH 3.5 buffer first, pH 6.5 buffer second, and pH 10.5 buffer last

In cation exchange chromatography: a. stationary phase is negatively charged and thus will bind negatively charged ions b. stationary phase is negatively charged and thus will bind positively charged ions c. stationary phase is positively charged and thus will bind negatively charged ions d. stationary phase is positively charged and thus will bind positively charged ions

b. stationary phase is negatively charged and thus will bind positively charged ions

The Beer-Lambert Law states that the fraction of the light absorbed is proportional to a. the spectrophotometer used and the temperature of the solution b. the time the sample i measured for and the path length c the path length and the concentration of the solution in which the light passes through d. the concentration of the solution and the spectrophotometer used

c the path length and the concentration of the solution in which the light passes through

HEPES has a pKa of 7.55. At what pH would a HEPES buffer have the maximum buffering capacity? a. 3.0 b. 4.76 c. 7.55 d. 8.00

c. 7.55

From the Lineweaver-Burk plot, which kinetic parameter can be determined from the y-intercept? a. Km b. Vo c. Vmax

c. Vmax

What is molality?

moles of solute/kg of solvent

"Parts" Dilution: What is 1:X mean?

-1 part stock: X parts diluent -ex. 1:1 = 1 part stock + 1 part diluent -ex. 1:9 = 1 part stock + 9 parts diluent

What is Volume by Volume OR % v/v?

-relative to volume of solution NOT volume of solvent -ex. 80% v/v CH3OH= 80 mL CH3OH in 20 mL H2O

What does precision mean?

-reproductibility -the degree by which repeated measurements yield similar results

What is weight by volume OR %w/v?

-weight is measured in grams -volume is measured in mL -ex. 5 %w/v NaCl= 5g NaCl in 100 mL H20

How do you calculate percent error?

(Iexperimental value- accepted valueI / accepted value) x100 **** ( ) is absolute value

"Parts" Dilution What is 1/X mean?

-1 part stock/X parts total -ex. 1/2 dilution= 1 part stock + 1 part diluent = 1:1 -ex. 1/10 dilution= 1 part stock + 9 parts diluent = 1:9

Which statement below is CORRECT with respect to SDS-PAGE? A larger protein will migrate faster in the gel than a smaller protein The gel has the same percentage of acrylamide throughout the entire gel After adding sample buffer to the protein sample, the proteins will have a net positive charge and migrate towards the negative electrode After adding sample buffer to the protein sample, the proteins will have a net negative charge and migrate towards the positive electrode

After adding sample buffer to the protein sample, the proteins will have a net negative charge and migrate towards the positive electrode

In lab this week, you will be given a sample containing a mixture of two proteins: cytochrome c (pI = 9.5) and ovalbumin (pI = 6.0). Cation exchange chromatography will be used to separate cytochrome c and ovalbumin. Which of the following statements below is CORRECT? At a pH of 6.5, cytochrome c will be positively charged and bind to the negatively charged cation exchange resin At a pH of 6.5, cytochrome c will be negatively charged and bind to the positively charged cation exchange resin At a pH of 6.5, ovalbumin will be positively charged and bind to the negatively charged cation exchange resin At a pH of 6.5, ovalbumin will be positively charged and bind to the positively charged cation exchange resin

At a pH of 6.5, cytochrome c will be positively charged and bind to the negatively charged cation exchange resin

What does micro mean?

Factor: 10^-6 OR .000001

What does deci mean?

Factor: 10^-1 OR .1

What does centi mean?

Factor: 10^-2 OR .01

What does milli mean?

Factor: 10^-3 OR .001

Given the information below, which of the following statements is CORRECT? Protein Molecular Weight (kDa) pI A 55 6.0 B 13 5.9 C 57 9.6 Given a mixture of Protein A and Protein B, ion exchange chromatography can be used to separate the two proteins Given a mixture of Protein A and Protein C, gel filtration chromatography can be used to separate the two proteins Given a mixture of Protein B and Protein C, gel filtration chromatography or ion exchange chromatography could be used to separate the two proteins Given a mixture of Protein B and Protein C, gel filtration chromatography can be used to separate the two proteins but ion exchange chromatography could NOT be used to separate the two proteins

Given a mixture of Protein B and Protein C, gel filtration chromatography or ion exchange chromatography could be used to separate the two proteins

You are given a mixture of the following two proteins: Protein 1 (MW 65kDa) and Protein 2 (MW 25kDa) Which statement below is CORRECT? If the sample is applied to a gel filtration column, you would predict Protein 2 to elute first If the sample is loaded in a well and separated via SDS-PAGE, you would predict Protein 1 to migrate faster If the sample is loaded in a well and separated via SDS-PAGE, you would predict Protein 2 to migrate faster If the sample is applied to a gel filtration column, you would predict Protein 1 to elute last

If the sample is loaded in a well and separated via SDS-PAGE, you would predict Protein 2 to migrate faster

Which of the following statements below are correct (select all statements that are correct!). SDS coats the protein backbone with a positive charge SDS coats protein backbone with a negative charge SDS linearizes the protein SDS removes secondary structure from proteins

SDS coats protein backbone with a negative charge SDS linearizes the protein SDS removes secondary structure from proteins

In the Separation of Proteins Part I lab, absorbance measurements will be used to monitor the elution of dyes and proteins off of the columns. Which statement below is CORRECT? The elution of cytochrome c and ovalbumin will be monitored at a wavelength of 640nm and the elution of blue dextran and vitamin B12 will be monitored at a wavelength of 400nm. The elution of cytochrome c and ovalbumin will be monitored at a wavelength of 400nm and the elution of blue dextran and vitamin B12 will be monitored at a wavelength of 640nm. The elution of cytochrome c, ovalbumin, blue dextran, and vitamin B12 will all be monitored at a wavelength of 640nm. The elution of cytochrome c will be monitored at a wavelength of 280nm and 400nm, the elution of ovalbumin will be monitored at a wavelength of 280nm, the elution of vitamin B12 will be monitored at a wavelength of 360nm, and the elution of blue dextran will be monitored at a wavelength of 640nm.

The elution of cytochrome c will be monitored at a wavelength of 280nm and 400nm, the elution of ovalbumin will be monitored at a wavelength of 280nm, the elution of vitamin B12 will be monitored at a wavelength of 360nm, and the elution of blue dextran will be monitored at a wavelength of 640nm.

When performing the Antioxidant lab, you will test the ability of substances to inhibit the______________ of nitro blue tetrazolium (NBT)? a. oxidation b. ionization c. hydroysis d. reduction

a. oxidation

In the alkaline phosphatase assay, you will you be monitoring the formation of product by measuring the absorbance of the solution using a spectrophotometer. Why are you monitoring the formation of product at a pH of 9 and a wavelength of 400nm. a. At pH 9, the product, p-nitrophenol, will be ionized, the solution will appear yellow in color, and thus can be monitored at the wavelength of maximum absorption for the phenolate ion which is 400nm. b. At pH 9, the product, p-nitrophenol, will be ionized and colorless c. At pH 9, the product, p-nitrophenol, will be un-ionized and yellow in color

a. At pH 9, the product, p-nitrophenol, will be ionized, the solution will appear yellow in color, and thus can be monitored at the wavelength of maximum absorption for the phenolate ion which is 400nm.

What type of inhibition is characterized by a change in Km but no change in Vmax? a. Competitive b. Noncompetitive c. Uncompetitive

a. Competitive

Why is EDTA used as the final step in the Western Blot procedure? a. EDTA is a chelating agent and will sequester metal ions and thus stop the alkaline phosphatase reaction b. EDTA enhances the colored product on the membrane c. EDTA is used to wash the membrane d. EDTA is an important component for the colorimetric reaction

a. EDTA is a chelating agent and will sequester metal ions and thus stop the alkaline phosphatase reaction

Which of the statements below is CORRECT? a. For the Superoxide Scavenging Assay, you will be using the 3mL cuvettes from your locker b. For the Superoxide Scavenging Assay, you will be using test tubes which can be directly inserted into the spectrophotometer. c. For the Superoxide Scavenging Assay, you will be provided with 1mL poystyrene cuvettes and for each assay performed, a new cuvette should be used d. For the Superoxide Scavenging Assay, you will be using the 1mL cuvettes from your locker

a. For the Superoxide Scavenging Assay, you will be using the 3mL cuvettes from your locker

At a ph of 9.5 what would you expect p-nitrophenol to be: a. ionized and yellow in color b. unionized and yellow in color c. ionized and colorless d. unionized and colorless

a. ionized and yellow in color

If you would like to determine the concentration of an Unknown solution using a standard curve and the absorbance reading of your Unknown sample falls outside the range of your standard curve (the absorbance reading of your Unknown solution is higher than the most concentrated standard): a. the standard curve should be extrapolated to determine the concentration of the unknwon b. the concentration of the unknown cannot be determines c. diluted samples of the unknown solution should be prepared so that the absorbance of the unknown solution is within the range of the standard curve. the standard curve can then be used to determine the concentration of the diluted unknown sample. In order to determine the actual concentration of the unknown solution, you must multiply the dilution factor that was used to prepare the diluted sample.

c. diluted samples of the unknown solution should be prepared so that the absorbance of the unknown solution is within the range of the standard curve. the standard curve can then be used to determine the concentration of the diluted unknown sample. In order to determine the actual concentration of the unknown solution, you must multiply the dilution factor that was used to prepare the diluted sample.

Given that the pI of aspartic acid is 2.77, at a pH of 8 aspartic acid will be: a.neutral b. positively charged c. negatively charged

c. negatively charged

What substrate will be used to assay the activity of alkaline phosphatase? a. phospate b. p-nitrophenol c. p-nitrophenyl phosophate d. phenol red

c. p-nitrophenyl phosophate

Which of the following statements below is NOT correct regarding thin layer chromatography: a. gloves must be worn when working with thin layer chromatography sheets b. samples must be spotted 2cm up from the bottom of the plate c. using pen write your initials d. samples must be spotted on the side coated with silica(not the shiny plastic side)

c. using pen write your initials

Which of the following statements below is correct: a. 40uL of 1N HCl must be added to the pH 6.5 fractions collected prior to spotting the fractions on the filter paper strip b. You do not need to save any of your fractions collected c. The amino acid sample should be applied to the column with force (push sample out of micropipettor as quickly as possible) d. 40uL of 1N HCl must be added to the pH 10.5 fractions, mix using the vortexer, and then spot fractions on the filter paper strips

d. 40uL of 1N HCl must be added to the pH 10.5 fractions, mix using the vortexer, and then spot fractions on the filter paper strips

Which of the following statements below is NOT correct? a. Affinity chromatography utilizes specific interactions between molecules b. Receptor-ligand binding is one type of interaction utilized in affinity chromatography c. Antibody-antigen binding is one type of interaction utilized in affinity chromatography d. Affinity chromatography separates proteins based on size

d. Affinity chromatography separates proteins based on size

Given that the pka of the caboxyl group of an amino acid is approximately 2.2, which of the following statements is correct: a. at a pH of 1, the carboxyl group would be deprotonated b. at a pH of 1, the carboxyl group would be negatively charged c. at a pH of 5, the carboxyl group would be positively charged d. at a pH of 5, the carboxyl group would be deprotonated and negatively charged

d. at a pH of 5, the carboxyl group would be deprotonated and negatively charged

The pH of a solution with [H+] concentration less than water would be a. 7 b. less than 7 c. acidic d. greater than 7

d. greater than 7

In lab you will be using affinity chromatography to purify lactate dehydrogenase from Lactobacillus acidophilus overexpressing lactatedehyrogenase. Which statement below is CORRECT. a. the lactate dehydrogenase was tagged with cyteine residues to facilitate the interaction with the Ni-NTA column resin b. the lactate dehydrogenase was tagged with glycine residues to facilitate the interaction with the Ni-NTA column resin c. the lactate dehyrogenase was tagged with alanine residues to facilitate the interaction with the Ni-NTA resin d. the lactate dehydrogenase was tagged with histidine residues to facilitate the interaction with the Ni-NTA column resin

d. the lactate dehydrogenase was tagged with histidine residues to facilitate the interaction with the Ni-NTA column resin

After proteins are transferred to the membrane, the membrane will be incubated in a blocking solution containing 10% milk. What does milk contain that is able to bind sites on the membrane that are not occupied by protein transferred from the SDS-PAGE gel? a. sugar b. water c. fat d. the protein casein

d. the protein casein

In lab this week, students will be using a spectrophotometer to measure the absorbance of various solutions. Which statement below is correct: a. the samples must be placed in the 1mL coveter from your locker prior to measuring the absorbance using the spectrophotometer b. a test tube can be directly inserted into the spectrophotometer c. the placements of the cuvette into the spectrophotometer is not important d. the samples must be placed in the 3mL cuvette from your locker and the cuvette must be placed into the spectrophotometer in a specific orientation to measure the absorbance

d. the samples must be placed in the 3mL cuvette from your locker and the cuvette must be placed into the spectrophotometer in a specific orientation to measure the absorbance

Gel filtration chromatography separates proteins based on____________________. charge charge and size polarity size

size

What does accuracy mean?

the degree of closeness of a measurement to its actual(true) value