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Malika Biochem MCAT > Methods of Separation & Analysis of Macromolecules > Flashcards

Methods of Separation & Analysis of Macromolecules Flashcards

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1
Q

Gel electrophoresis separates proteins based on their _______ and ________

A

net charge and size

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2
Q

Do positively charged amino acids flow towards the anode or cathode in electrophoresis?

A

Cathode

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3
Q

In gel electrophoresis, the anode is ______ charged and the cathode is ______ charged.

A

positively, negatively

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4
Q

Gel electrophoresis can also be used to separate DNA by size. DNA which has 500 bp travels (faster/slower) than DNA with 165 bp.

A

slower

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5
Q

There are 2 types of gel electrophoresis for running DNA, what is the difference between them?

A

Polyacrylamide gel electrophoresis (PAGE) - separates short DNA fragments
Agarose gel electrophoresis - separates longer DNA fragments

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6
Q

How do you get better resolution of bands?

A
  1. Running the gel at a lower voltage for a longer period of time
  2. Using a wider/thinner gel comb
  3. Load less DNA into the well
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7
Q

How do you get better separation of bands on an agarose?

A

Adjust agarose % of the gel.
1. For separating small bands - need higher % agarose gel
2. For separating larger bands - need lower % agarose gel

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8
Q

What is the purpose of ethidium bromide when running a gel?

A

It binds to DNA and allows you to visualize DNA under UV light.

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9
Q

What is native PAGE?

A

Native polyacrylamide gel electrophoresis. Analyzes proteins in their native folded states (size, mass & charge)

*Consider the charge/mass ratio
*Consider bigger and less compactly folded proteins travel slower

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10
Q

What is SDS-PAGE?

A

Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Technique that separates protein based on their molecular mass ONLY.
Proteins are given UNIFORM negative charge.

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11
Q

The two types of SDS page are reducing and nonreducing: define them

A

Reducing - breaks up covalent disulfide bonds
Nonreducing - doesn’t break up disulfide bonds, but still breaks other intramolecular bonds

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12
Q

What is the reducing agent used to separate disulfide bonds?

A

beta-mercaptoethanol

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13
Q

How protein proteolysis vs protein aggregation presents on a gel

A

A - protein aggregation bc bands get thicker at the end, meaning they have combined
B - protein proteolysis because proteins get separated into smaller bands

*The amount of protein over time across the row decreases as the experiment goes on. These are not loading wells, (if they were, they would have equal bands)

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14
Q

What is isoelectric focusing?

A

A technique utilized to separate proteins based on their isoelectric point. A gel with a pH gradient is created with acidic gel at the positive anode side and basic gel at the negative cathode side. Once proteins migrate to the corresponding side of opposite charge, they will stop moving when they reach the portion of gel where the protein’s pI = gel’s pH.

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15
Q

In isoelectric focusing, the anode side has ______ gel, while the cathode side has ______ gel.

A

acidic, basic

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16
Q

In chromatography, is the stationary phase nonpolar or polar?

A

Polar

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17
Q

What is thin layer chromatography (TLC)?

A

A technique which separates compounds based on polarity utilizing a thin glass plate coated with silica gel as the stationary phase and a liquid solvent as the mobile phase.

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18
Q

In TLC chromatography, if the compound travels a larger distance, is it more polar or more nonpolar?

A

More nonpolar because it has low affinity for the polar mobile phase, so it wants to escape more quickly.

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19
Q

In TLC chromatography, compounds that have high affinity for the stationary phase will migrate more (quickly or slowly)?

A

More slowly

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20
Q

In chromatography, what does affinity mean?

A

Affinity is determined by how alike the mobile phase is to the stationary phase.

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21
Q

True or false?:
In TLC chromatography, higher the retention factor, the less polar the compound is and the more quickly it travels.

A

True

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22
Q

How do you determine retention factor?

A

You divide the distance traveled by the compound of interest/distance traveled by solvent front. The solvent front is the baseline at the end of the thin paper.

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23
Q

What is column chromatography?

A

A column is filled with polar silica or alumina beads (stationary phase) and a solvent (mobile phase) is poured into the column. Molecules that are less polar elute first. Molecules that are more polar interact with the beads and therefore elute slowly.

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24
Q

What are some types of column chromatography other than the standard one measuring polarity?

A

Ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography

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25
Q

What chromatography is this?:
Beads contain tiny pores which retain molecules that are smaller and allow bigger molecules to flow through.

A

Size-exclusion chromatography

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26
Q

What type of chromatography is this?:

Beads in the column are customized by coating them with a receptor or antibody specific to the protein of interest that we want to isolate.

A

Affinity chromatography

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27
Q

What is ion-exchange chromatography?

A

Beads in the column are coated with a charged substance so they attract the compound with the opposite charge, isolating it.

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28
Q

If the beads in the column are coated with a positively charged substance, is it cation or anion exchange chromatography?

A

It is anion-exchange chromatography.

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29
Q

If the beads in the column are coated with a negatively charged substance, is it cation or anion exchange chromatography

A

It is cation-exchange chromatography.

30
Q

Special type of affinity chromatography:
His-tag affinity chromatography

A
  1. In first step the polyhistidine tag of the recombinant protein binds to the Ni2+ cations on the column
  2. Then another molecule which mimics the side chain of histidine such as imidazole is added to the column to elute the histidine proteins.
31
Q

Explain the process of gas liquid chromatography

A

A gas mobile phase is injected into a heated chamber (inert N2 gas) causing the mobile phase to vaporize and then it is pushed through a long coiled tube (coated with liquid stationary phase) which connects to a chromatogram detector at the end. The detector graphs peaks of which compound elutes first.

32
Q

Gas-liquid chromatography separates compounds based on what two factors?

A

Most importantly boiling point and molecular weight
-Compounds which have a low BP elute first

33
Q

What is distillation used for?

A

It is used to separate compounds in a mixture based on boiling points.

34
Q

Explain the process of simple distillation.

A

The distilling flask with the mixture is placed in a hot oil bath. It is connected to a thermometer and an adaptor with a vacuum which connects to the receiving flask in the ice bath. As a compound vaporizes it moves up the adaptor condensing several times and moved through the vacuum eventually dripping into the receiving flask.

35
Q

What is the purpose of the vacuum in distillation?

A

It’s purpose is to lower the atmospheric pressure, which allows a substance to reduce its boiling point and condense into the receiving flask quicker.

36
Q

Vacuum distillation is ideal for what kind of compounds?

A

Compounds that have boiling points over 150C to prevent degradation.

37
Q

How is fractional distillation different from simple distillation?

A

In fractional distillation, you have a fractionating column that is filled with glass beads/packaging material which allows the substance in the compound mixture to vaporize and condense several times in the column before reaching the receiving flask. Essentially, producing a much purer substance.

38
Q

Explain the technique of extraction

A

It is used to selectively remove a compound of interest from a mixture. By taking advantage of the different solubilities of the compounds in the mixture, a compound can be separated into the aqueous phase once it is either protonated/deprotonated.

39
Q

How would increasing the length of the column in fractional distillation affect the separation of compounds?

A

It would help to increase the separation of compounds, causing them to be more pure.

40
Q

What is the purpose of boiling chips or a stir bar?

A

They are used to prevent superheating and ensure even heating.

41
Q

In extraction, which phase is the hydrophobic layer?

A

Organic phase

42
Q

In extraction which phase is the hydrophilic layer?

A

Aqueous phase

43
Q

In the aqueous layer, molecules exist in their (neutral/charged) form.
In the organic layer, molecules exist in their (neutral/charged) form.

A

Charged, neutral

44
Q

If you need to extract a molecule w/ acidic properties, what solution can you use?

A

Extracting:
Strong acid (carboxylic acid) = can use mild base NaHCO3 or strong base NaOH-
Weak acid (phenol) = use ONLY strong base NaOH

45
Q

What solution can you use to extract an amine?

A

A strong acid such as HCl or H2SO4 will cause the amine to go to the aqeuous layer

46
Q

Example SB C/P #12

A
  1. Add 0.1 M NaOH to quench unreacted acid anhydride. Then, add diethyl ether to separate the layers. The amide can be obtained from the ether layer by evaporating the solvent.

*Adding NaOH first allows it to react with the acid anhydride to neutralize it, and separate it into the aqueous layer. The amide is left behind in the organic layer & can be evaporated out.

47
Q

What are common organic solvents used in extraction (which make up the organic layer of the separatory funnel)? What about common solvent utilized for the aqueous layer?

A

Organic - Ethyl acetate, diethyl ether, chloroform, dichloromethane, n-hexane
*They all have different densities
Aqueous - methanol, ethanol, maybe ethyl acetate (slightly polar but still works as an organic solvent)

48
Q

What is X-ray crystallography used for?

A

This technique allows for visualization of tertiary protein structure.

49
Q

Explain the process of X-ray crystallography

A
  1. The protein has to be in its crystallized form.
  2. The protein crystal is bombarded with a beam of x-rays and the radiation scattered by the electrons of the proteins strikes an electron sensitive detector
  3. The pattern of diffraction produced shows the structure of the protein
50
Q

What are some disadvantages of x-ray crystallography?

A
  1. It is harder to analyze complex multi subunit membrane proteins with this technique.
  2. The protein has to be able to be crystallized.
51
Q

What is NP (normal phase) high performance liquid column chromatography?

A

A type of column chromatography where a pressure pump is placed at the top of the column which pumps the liquid mobile phase into the column at a high pressure. The column is filled with beads that interact with the mobile phase and allow separation of compounds from the mixture. The elution of the compound can be recorded on a chromatogram. The # of peaks correspond to how many different molecules there are in the mixture.

52
Q

Longer retention time on an HPLC chromatogram indicates (greater/lesser) affinity for the mobile phase.

A

lesser affinity for mobile phase, BUT greater affinity for stationary phase

53
Q

Can HPLC be used to determine the purity of a reaction mixture?

A

Yes, depending on the # of peaks recorded on the chromatogram, HPLC can be used to determine whether a mixture is pure or not, and how many different compounds are in it.

54
Q

HPLC measures (polarity/boiling point) of compounds.

A

Polarity

55
Q

Reverse HPLC uses a (polar/nonpolar) stationary phase and a (polar/nonpolar) mobile phase.

A

Nonpolar stationary phase, polar mobile phase

56
Q

Small molecules such as individual amino acids & dipeptides are be separated via ____________, whereas large molecules like proteins are separated via ______________.

A

HPLC, gel electrophoresis

57
Q

What is paper chromatography?

A

Usually the fastest and cheapest method of particle separation that uses a cellulose filter paper as the stationary phase and a liquid solvent as the mobile phase. Efficient for separation of water soluble, polar compounds.

58
Q

Uses of recombinant DNA technology

A
  1. Isolation, identification , mapping and sequencing of genes.
  2. Describing the function of a gene.
  3. Production of recombinant proteins for lab experiments.
  4. Development of drug delivery systems.
59
Q

What is the purpose of chiral column chromatography?

A

This is a type of column chromatography that allows for the separation of enantiomers with the use of a chiral stationary phase. One of the enantiomers interact more closely with the stationary phase which is designed to bind either the R or S conformation.

60
Q

Reverse transcriptase-PCR

A

Converts mRNA into cDNA to then use to amplify and create many cDNA sequences
-Used to create a cDNA library
-Used to analyze gene expression
-Used to detect if a certain RNA is present (say from a virus?)
Process:
-Uses mRNA as a template and random hexamer primers

61
Q

Real-time PCR

A

-Measures fluorescence of the DNA/RNA created in real time PCR
-The fluorescence produces a curve & whichever is steepest, produces
more of the sequence over time
-Used to measure AMOUNT of sequence present

62
Q

Fractional distillation is ideal for liquids with a boiling point difference of _______.

A

Less than 25 degrees C. Fractional distillation provides a larger surface area for vapors to travel before condensing, which allows for a more pure compound separation.

63
Q

Steps of polymerase chain reaction (PCR).

A
  1. DNA is heated to 90°C to separate the two strands.
  2. The DNA is quickly cooled to 30C—65°C, which allows for the short single strand primers to anneal to their complementary sequences.
  3. The solution is then heated to 70°C and DNA polymerase is added. The synthesis of two new double stranded DNA molecules occurs.
  4. The cycle is then repeated. Each time the cycle repeats, the amount of target DNA doubles.
64
Q

If you go through 20 rounds of PCR, how many DNA molecules do you have?

A

2^20 power

65
Q

Requirements for creating primers for PCR:

A
  1. 40-60% GC content
  2. Complementary to template region of DNA at the 5’ and 3’ end
  3. Start & end w/ 1-2 G/C pairs
66
Q

Sanger Sequencing

A

Purpose: Determines the sequence of a DNA strand, fast speed in smaller samples
Need: dNTPs, DNA template strand, DNA polymerase, ddNTPs, primers
How to read the gel: Start all the way at the bottom at the ddTTP lane and work your way up from each band that is smallest all the way to largest band

Key: When a ddNTP is added to the strand, no new dNTPs can be added, thus terminating the strand at a random point and allow for multiple size fragments to be generated, a fluorescent label is placed on the ddNTP to visualize it

67
Q

ELISA (enzyme-linked immunosorbant assay)

A

Purpose: Method which utilizes a primary & secondary antibody to determine & quantify the presence of a specific protein of interest
Indirect ELISA - The # of signals on the well tells you about the concentration of antibody of interest
–The secondary antibody binds directly to the primary antibody
Sandwich ELISA - Used to detect the presence of an antigen (quantity of antigen)
–The secondary antibody binds directly to the antigen

68
Q

Compare the 2 types of immunofluorescence (IF): immunohistochemistry (IHC) & immunocytochemistry (IC)

A

Overall purpose: Most importantly, localization of protein ,and secondly expression of protein (but Western Blot is superior for expression levels)
IHC - used in tissues
IC - used in cells

69
Q

Direct vs. indirect immunofluorescence

A

Direct - uses only a primary antibody with a conjugated fluorophore
Indirect - uses a primary antibody & secondary antibody w/ a conjugated fluorophore resulting in amplification of signal

70
Q

Fluorescence In Situ Hybridization (FISH)

A

Technique that allows the localization of a specific DNA sequence on a chromosome or (deletion) of genes on a chromosome
1. Design a probe that has fluorophore labeled nucleotides and matches your gene of interest
2. Allow probe to hybridize with your gene of interest in the cell chromosome
3. If probe doesn’t hybridize (no fluorescence detected), that means gene is deleted on the chromosome

Malika Biochem MCAT (11 decks)

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