Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Biochem II Self Study > Biochem lab final exam > Flashcards

Biochem lab final exam Flashcards

1
Q

You have an interview in two weeks, which will result in your missing a lab or lecture. To whom should you address your email asking for accommodations?

A

All emails, unless they involve confidential content are to sent to the instructor, your GTA, and all four UTAs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is the gene that encodes for a barracuda striated muscle LDH monomer?

A

LDHA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the applications for the following four spectrophotometer wavelength (nm): 260, 280, 340, and 595?

A

Used for DNA detection (260), Protein detection (280), NADH detection (340), and Bradford assay (595)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

In the equation, A = εlc, what does each of the variable stand for?

A

They stand for absorbance (A),

the extinction coefficient (ε),

the path length (l)

the protein concentration (c)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Give the official isozyme name for beef heart LDH, the name and number of monomers that comprise the completed protein, and the gene that encodes for those monomers.

A

Beef heart LDH (LDH-1) is made up of our LDH-H monomers, which are each encoded for by the LDHB gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

You cannot directly measure the activity of a protein, but rather need to measure a byproduct of its reaction. What is it that you are measuring in the activity assay for LDH and what wavelength do you monitor?

A

The LDH activity assay is monitoring the formation of NADH (not NAD+) and is monitored at 340 nm.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the two restriction enzymes that we are using for the preparation of our expression vector?

A

We will be using BAMH1 and NDE1 for the digestion of our DNA in preparation of our expression vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

When making a buffer, why is it important to pH our buffer prior to bringing it up to the final volume?

A

If you pH the buffer after bringing it to the final volume, your calculated concentration values will be off from their actual values

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

In this week’s chromatography method, supports (also known as resins) are used to immobilize what?

A

Supports immobilize the “exchanger charged groups”

which in turn, immobilize the protein

(chromatography method - IEC)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

IEX and IEC are both abbreviations for what chromatography method?

A

IEX and IEC both refer to “Ion Exchange chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

With this week’s chromatography method, the protein of interest must “stick to” the column. What is it about the protein that makes it “sticky”?

A

Charged and/or polar residues on the protein’s surface “stick” to the column’s exchanger charged groups by way of electrostatic interactions

IEC

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

If your protein of interest (for today’s chromatography method) had predominately negative surface charge, what type of IEC column would you want to use?

A

Use an Anion Exchange (immobilized cation) column to attract the protein’s negative surface charge

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

You want to make 75 mL of a 20 mM buffer that has a molecular weight of 100 g/mol. Show your work for how you would calculate the grams of solute needed for this buffer.

A

0.02 mols/L * 100 g/mol = grams of solute per liter of
solvent

g/L = mg/mL

mg/mL * 75 mL = mg of solute needed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Last week you took A280 and activity measurements of the fractions that eluted off the IEC column. How could you have an A280 measurement of 0.7 with a negligible slope on your activity assay?

A

As there is still a large quantity of extraneous protein in our sample that elute off the IEX column at different times/buffer compositions, you can have an absorbance of 0.7 at 280nm without having activity as 280 nm is a measurement generic to protein not specific to LDH

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is the major difference between IEC and Affinity chromatography’s?

A

IEX column separates proteins based on charge while affinity chromatography separates proteins based on their affinity to the immobilized ligand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

In this week’s lab, what molecule is the mimic mimicking

A

Cibacron Blue is a molecular mimic of NADH, a cofactor of LDH enzyme

AFC - affinity chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is being quantified with the Bradford assay?

A

All proteins are are being quantified. While we hope that the sample is getting “pretty pure” there are still other proteins in there

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

The ligation reaction that attaches the DNA insert into the vector DNA is endergonic and therefore requires what molecule?

A

ATP to provide reaction energy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What is the word used to describe the technique used by scientists in which they incorporate a plasmid into bacteria?

A

Transformation/transfection are terms used to describe the uptake of external DNA by bacterial vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What dye is used in the bradford assay, and what is the dye’s bound absorbance wavelength? (extra credit for dye’s unbound absorbance wavelength)

A

The dye, Coomassie Brilliant Blue (CBB), aka Bradford reagent, absorbed at 595 nm when bound and 470 when not bound

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is the lab process called whereby bacteria take up DNA that they did not previously have?

A

Transformation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What ingredient in this lab’s plates kills some bacteria, only allowing those bacteria that successfuly took up the new DNA plasmids to live and reproduce (selection)?

A

Ampicillin is added to the plates to select for bacteria that successfully took up the DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What is the name of the covalent bonds that are formed during a vector + ligation reaction?

A

these are called phosphodiester bonds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

How many of these covalent bonds are required for one individual vector + insert ligation?

A

Four bonds are required; the DNA has 2 strands and 2 ends, that 4 bonding locations

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What is the procedure for which you are choosing a colony in this week’s lab?

A

we are choosing colonies for PCR (polymerase chain reaction)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Heart LDH

A

Isozyme name: LDH-1

Gene encoding for a monomer: LDHB

How many monomers: 4

Name of monomer: LDH-H

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Striated Muscle LDH

A

Isozyme name: LDH-5

Gene encoding for a monomer: LDHA

How many monomers: 4

Name of monomer: LDH-M

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

From Adam Smiley’s lecture, what company is the “winners’ of NGS”

A

Illumina is Adam’s choice for excellence (the “winners”) in the current next generation sequencing technology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

What metal is used for our IMAC protocol this week?

A

Nickel (Ni2+) is the metal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

A short sequence of amino acids was added to our expressed LDH-M polypeptide sequence to cause it to stick to the IMAC column.

A

The n-terminally attached “His-tag” sequence is comprised of 6 histidines in a row

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Ultimately, the immobilized protein will need to be eluted off of the column. What chemical is added to outcompete the protein’s affinity to stick to the column?

A

Imidazole in solution outcompetes and disconnects the His-Nickel binding, and causes the protein to elute off

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

Why not just use an SDS-PAGE or polyacrylamide gel on our proteins? What advantage does a western blot have over a more conventional gel?

A

Conventional gels separate proteins based on molecular weight. Other proteins could have the same MW as LDH.

Western blot will isolate our specific protein based on exclusive antibody binding

33
Q

Southern blots, northern blots, western blots… Which of these is actually names after a person?

A

Southern blots are named after a person (Edwin Southern)

34
Q

Blots and what they display?

A

Southern blots display DNA
Northern blots are for RNA
Western blots are for protein

35
Q

What protein(s) will we be western blotting in this week’s lab?

A

Will be western blotting both (muscle LDH and beef heart LDH)

36
Q

Why is protein transferred onto a membrane necessary in western blotting?

A

Protein transfer onto a membrane is necessary to immobilize the proteins from the gel.

The membrane provides a solid support where proteins maintain their spatial arrangement, facilitating subsequent antibody binding and detection

37
Q

What do the secondary antibodies bind to in western blotting?

A

Secondary antibodies recognize and bind to the primary antibodies

38
Q

What is the purpose of blocking with milk in western blotting?

A

To prevent nonspecific binding of antibodies to the membrane. This step reduces the background signal and improves the specificity of protein detection

39
Q

What specific reactions are used for determine the presence of different forms of LDH?

A

HRP (horseradish peroxidase) reaction for barracuda

AP (alkaline phosphatase) reaction for beef heart

40
Q

The gels you have run prior to this lab are very different from the gel in this week’s lab in what ways?

A

In this lab, the method (native gel)

  1. does not denature the protein
  2. does not destroy the enzyme’s activity
  3. allows for an in-gel assay to be performed
  4. observed the tetrameric for on a gel (first time)
41
Q

For this week’s lab, the reaction used to detect the forms of LDH is run on what gel material?

A

This week’s gel (native gel) is polyacrylamide (not agarose).

42
Q

lactate –> _____, drives the rxn

A

pyruvate

43
Q

____ –> NADH, drives rxn

A

NAD+

44
Q

phenazine (oxidized) –> ______, drives

nitroblue —> _____ (purple)

A

phenazine (reduced)

formazan

45
Q

Which will travel further during gel electrophoresis, a heavier DNA sample or a lighter DNA sample?

A

A lighter DNA sample would travel further during gel electrophoresis. The lighter DNA samples face less resistance going through the gel and then will go further than the heavier samples.

46
Q

You are trying to determine the extinction coefficient of a compound. The absorbance is measured at 0.495. The concentration of the sample is 0.03 M and the path length is 1 cm.

A

A = ε * c * l

0.495 nm = ε * (0.03 M) * (1 cm)

ε = 16.5 M-1cm-1

47
Q

What is the purpose of Nde1 and BamH1?

A

They are restriction enzymes

48
Q

How would your results have been affected if you neglected to change
wavelengths between Bradford or Kinetics Assay? What wavelengths are used for each
assay?

A

The wavelength used for the Bradford assay was 595 nm and the wavelength used for the Kinetics Assay was 340 nm. If we neglected to change the wavelengths between the two assays it wouldn’t allow us to read the absorptions for the right components. NADH has a specific peak around 340 nm and Bradford dye can be read at 595 nm.

49
Q

If you see a procedure that leads to a decrease in specific activity of LDH, what are two possible reasons?

A

A possible decrease in LDH activity would be due to the denature of the enzyme by harmful conditions. Such as the wrong temperature of storage during the experiment.

Another possible reason is that there was a large delay from inputting the enzyme and recording the activity.

50
Q

What is significant about a 65% salt concentration for the purification process?

A

it is where LDH is no longer soluble and will precipitate.

51
Q

Activity (µmol/min)

A

Abs/sec * 60sec/min = abs/min

Abs/6220 = M/min

M/min = mol/L*min * 0.001 L = mol/min

mol/min * 10^6µmol/mol = µmol/min

52
Q

Total units (Units)

A

U/ml * mL (total sample) = Units

52
Q

relative activity (U/ml)

A

activity/sample added

0.161U/0.01mL = U/mL

53
Q

Percent recovery

A

Percent recovery = (total units of fraction/total units of crude) X 100

54
Q

What was the purpose of performing a dialysis on the sample (65% pellet)?

A

The purpose of performing adialysis on the sample is to remove the salt (AmSO4) from the solution while retaining the protein

55
Q

What is initial velocity and why is it important when working with enzyme kinetics?

A

Initial velocity is the the initial rate of reaction of enzyme and substrate. It is important in enzyme kinetics because the reaction rate will eventually slow to zero as the substrate is used up

56
Q

why is it necessary to balance the centrifuge when spinning your samples?

A

if not balanced it will damage the centrifuge

57
Q

What 4 components are being “cleaned-up” or removed from your PCR samples during PCR cleanup? What is the importance of removing these components?

A

enzyme (DNA pol)

components that are being “cleaned-up” from the PCR samples during PCR
clean up are salts, dNTPs, primers, and other impurities. It is important to remove these
components because it could interfere with the analysis of our desired protein, LDH.

58
Q

How would you make 200 ml of a 0.1 M stock solution X of a substance that has
a molecular weight of 121.1 g/mol?

A

X/MW /L = M

X/ mol/g = mol

X = g

59
Q

In research labs, the most common chromatography methods are ion exchange, affinity, and size exclusion. What property of the protein of interest does each method rely on?

A

The property of protein of interest ion exchange relies on the charge of the protein.

The property of the protein of interest that affinity chromatography relies on is the ligand binding affinity of the protein.

The property of protein of interest size exclusion relies on is the molecular weight and size of the protein

60
Q

You’re setting up a purification for beef heart LDH-H and the only ion exchanger available is sulpho-styrene. Is this a good fit for purifying LDH? Why?

A

Sulpho-styrene is a strong cation exchanger and is not a good fit for purifying LDH because LDH has an overall negative charge and would need an ion exchanger that has a positive charge

61
Q

What amino acids do NADH and pyruvate bind to in the LDH enzyme? What kinds of bonds do they use?

A

NADH and pyruvate are shown bound to the active site of LDH. The binding is through ionic interaction and hydrogen bonding with tyrosine, lysine, histidine, aspartate, and arginine amino acids

62
Q

In this lab we only used 2 buffers, yet a variety of solutions with different concentrations were made during the FPLC (DuoFlow). How was this accomplished?

A

This was accomplished by the FPLC machine imputing the different concentrations in the column. There are two buffers that are pumped into the FPLC to achieve the different concentrations.

63
Q

Specific Activity

A

relative activity/protein concentration = U/mg

64
Q

What are the components needed for our ligation reaction to occur?

A

DNA fragments (insert + plasmid, both cut) and ATP/buffer.

65
Q

On a molecular level, how is ligation accomplished and what is the name of the chemical bond that is formed?

A

Ligation is accomplished by the joining of DNA fragments with compatible cohesive ends using the DNA ligase enzyme. It catalyzes the formation of the phosphodiester bond between the 3’ hydroxyl end of one DNA fragment and the 5’ phosphate end of another

66
Q

Suppose you have a circular plasmid that contains one restriction site. If the restriction enzyme is present how many bands would you expect on your gel?

A

You would expect one band for the linearized plasmid and one for the supercoiled form.

67
Q

Enzyme 1 has a KM of 0.2 M. Enzyme 2 has a KM of 5 M. Which enzyme has a
stronger affinity for the substrate?

A

The Michaelis constant KM is a measure of the affinity of an enzyme for its substrate.

A lower Km indicates higher affinity for the substrate.

Therefore, enzyme 1 has a stronger affinity for the substrate because it has a lower Km value than Enzyme 2.

68
Q

What values can be derived from the intercepts on the Lineweaver-Burk plot?

A

The intercepts are 1/Vmax for the Y intercept and the X intercept is -1/Km.

69
Q

What is the purpose of performing single colony PCR on your bacterial sample? What are you amplifying in this sample?

A

The purpose is to check if the ligation reaction was successful. We don’t need to amplify our LDHA gene any further, it is purely a check to see if it worked before we express. It also has nothing to do with purity.

70
Q

If you found no results in your PCR, what may have happened to your bacteria?

A

Transformation is the taking up of a vector. we know it took one up because it lived on the AMP plate, it means the ligation reaction didn’t work

71
Q

What is different regarding some of the nucleotides in Sanger Sequencing? Why
do we add them?

A

In Sanger sequencing we use a special type of nucleotide called a dideoxynucleotide
(ddNTP).

The difference between a normal deoxynucleotide (dNTP) and a dideoxynucleotide is
that ddNTP lacks a 3’-OH group on the sugar ring.

Each base is also tagged with different fluorescent labels. These labeled nucleotides terminate DNA synthesis, allowing determination
of the nucleotide sequence.

72
Q

What type of chromatography is IMAC?

A

type of affinity chromatography

73
Q

What is the functional group present on the side chain of histidine residues that causes our His tag to bind to the Ni-NTA resin?

A

the functional group is the imidazole group present in the side chain of histidine residues

74
Q

A certain compound is present at a high concentration (500mM) in the elution buffer. What is said compound and what is its purpose in the elution buffer?

A

The compound present at a high concentration (500mM) in the elution buffer is imidazole.

Imidazole competes with histidine residues of the His-tagged protein for binding to the Ni-NTA resin, facilitating the elution of the protein from the resin

75
Q

One of your colony PCR samples showed no band on the agarose gel. What could this indicate about the colony you picked for this specific sample?

A

this failure is only due to lack of ligation

76
Q

What does the SDS buffer do to proteins?

A

SDS (Sodium Dodecyl Sulfate) buffer denatures proteins by binding to them and disrupting their structure. It also gives a negative charge to proteins and facilitates their separation based on size during gel electrophoresis.

77
Q

How can you determine the purity of a sample via an SDS-PAGE gel?

A

Purity is seen through number of bands in a lane. more bands = less pure

78
Q

What does “PAGE” stand for?

A

polyacrylamide gel electrophoresis

Biochem II Self Study (10 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions