MCAT Lab Techniques

Question 1

What is the purpose of gel electrophoresis?

Answer 1

Uses an electric field applied through a gel which separates proteins.

The gel can be agarose ( a complex carb) or polyacrylamide ( organic polymer).

Question 2

Do biomolecules with a higher magnitude of charge travel slower or faster through the gel?

Answer 2

The biomolecule with higher magnitude travels faster through gel because of it interacts more strongly with the electric field.

Question 3

Describe the difference between gels used in gel electrophoresis?

Answer 3

Agarose gel (a complex carbohydrate) has larger pores and so it used to separate DNA and RNA.

Polyacrylamide gel ( an organic polymer) is smaller pores that are good for proteins.

Question 4

Describe the process of gel electrophoresis?

Answer 4

The gel is an electrolytic cell with a positive anode and a negative cathode with the charge moving from the cathode to the anode.

We place biomolecules into gel. Depending on their charge they move through the gel until the electric field is stopped.

We stain the biomolecules with a coomassie gel which stains them blue to allow them to stand out and be visualized.

• Smallest molecules and those with a large charge migrate the fastest.
• Thicker bands means more of the biomolecule while thinner bands means less.
• Bands further down the gel migrated faster and therefore weigh less than the biomolecules at the top.

Question 5

What is the ladder in gel electrophoresis?

Answer 5

A lane with a biomolecule of known charge and size and is used to estimate the sizes the biomolecules we’re testing in the gel.

Question 6

Native electrophoresis
( Native- PAGE)

Answer 6

Separation of biomolecules without modification to the structure.

Usually used with DNA and RNA because naturally have a negative charge.

Question 7

Describe the reason for Native- PAGE?

Answer 7

Because we don’t disrupt the structure of the biomolecule we can retain it’s structure. Because protein function is preserved it we can use the biomolecule after it’s separation.

• Proteins with a low PI has a negative charge
• Proteins with a high PI has a positive charge.

Question 8

Migration shift

Answer 8

When the protein captured from Native-PAGE interacts with it’s ligand making it heavier and alters it’s movement in the gel.

Question 9

SDS- PAGE

Answer 9

Uses a denaturing agent called sodium dodecyl sulfate which denatures the protein and surrounds it with a negative charge.

This eliminates charge as a factor in movement but protein function is disrupted.

SDS is NOT a reducing agent therefore it doesn’t break apart disulfide bonds.

Question 10

How can we find the mass of a protein?

Answer 10

By using the mass of an individual amino acid which is 110Da ( dalton) = 1000 KDa

Question 11

What happens with a “homo” and “hetero” protein?

Answer 11

If a protein has the same size subunits ( homo) they all appear as one band that appears at the same distance.

If the proteins has different size subunits ( hetero) they all appear as different bands at different distances.

Question 12

What happens when we subject a protein with disulfide bonds to a nonreducing SDS-PAGE?

Answer 12

The disulfide bonds will not be broken therefore you will a see a single band.

Question 13

How are disulfide bonds are created?

Answer 13

oxidation of cysteine residues to cystine residues.

Reversing this is done by adding a reducing agent by breaking apart the cystine bond going back to a cysteine bond.

Question 14

What are common reducing agents?

Answer 14

DTT ( Dithiotherital)

BME ( beta- mercaptoethanol)

Question 15

Describe the theory of isoelectronic focusing?

Answer 15

Separates proteins based on PI.

Contains an electrolytic cell with a polyacrymalide gel combined with a pH gradient.
- Anode = low pH = high H+ concentration.
- Cathode = high pH = low H+ concentration.

• proteins in which the pI> pH the protein picks up protons.
• Proteins in which the pI < pH it loses protons.

Question 16

What dictates the movement of proteins in IEF?

Answer 16

Protein with a positive charge moves the negative cathode side.

Protein with a negative charge moves to the positive anode side.

Migration stops when the pI = pH.

Question 17

2D technique electrophoresis

Answer 17

Technique to further protein separation.

First we use IEF and then immediately SDS- PAGE.

Creates data with molecular weight on the y-axis and IEF on the x-axis.

Question 18

Describe how blotting works?

Answer 18

We transfer proteins that has been separated via gel electrophoresis (which contains desired and undesired biomolecule).

We attach the biomolecules to the membrane and add a probe that specifically binds to the biomolecule of interest.
- For nucleic acids we use single- stranded DNA
- For proteins we use antibodies

Question 19

What is blocking in blotting techniques? What’s the purpose?

Answer 19

Blocking is used to prevent probes from attaching to the membrane itself in addition to the biomolecule of interest.

We saturate the membrane in a solution that prevents the probes from binding to the membrane.

Question 20

What are the ways we use probes for visualization during blotting techniques?

Answer 20

We can use probe directly labeled in which the probe is attached directly to the primary antibody, or we can use probe indirectly labeled in which the probe is attached to a secondary probe which binds to the primary probe.

Probe provides a visualization when it undergoes radioactive decay. Signals appear as bands on the film.

Question 21

Autoradiography

Answer 21

The process of detecting radioactive labels using radiographic films.

Question 22

How can we determine if a proper amount of biomolecule is loaded onto the blotting membrane?

Answer 22

We can quantify the amount or use the labeling of housekeeping genes (genes always expressed in every cell).

This is important because we want to make sure that changes, we observe is not due to loading errors but to experimental conditions.

Question 23

Dot blot

Answer 23

A blotting technique that doesn’t separate proteins via gel electrophoresis first.

Question 24

Why do we perform gel electrophoresis first before blotting in most instances?

Answer 24

To separate the component of interest from the other components that are in the cell lysate. We add all the separated proteins (proteins were interested in and not interested in) so we can label and identify the one we’re interested in.

Question 25

What is the purpose of blotting?

Answer 25

To identify a biomolecule of interest from a cell lysate that we separated using gel electrophoresis.

Question 26

Southern blots

Answer 26

Used to identify DNA. Gel electrophoresis is performed first to separate the strands based on size. We then denature them in basic conditions so we can hybridize probes to them.

We transfer the samples from the agarose gel to the blotting membrane via upward capillary transfer.

Probes used to identify is SSDNA and unbound SSDNA can be washed so bands can be visualized.

Question 27

Describe the process of upward capillary transfer

Answer 27

Used to separate DNA from gel to blotting membrane for southern blotting.

Steps:
1. blotting membrane is placed on top of the gel.

2. membrane- gel pair is placed in- between two filters. The bottom filter is placed on top of a buffer that’s in a reservoir.
3. We place paper towels on top of the top filter paper and place a weight on top of that.
4. The buffer moves up via capillary action transferring the DNA from the gel to the membrane.

Question 28

Nothern blots

Answer 28

Used to separate RNA. The same process as southern blots with two exceptions:

1. RNA can have internal base pairing, so they need to still be denatured.
2. We cannot denature RNA under basic conditions so we must do so using formaldehyde.

Question 29

Western blots

Answer 29

Blotting technique that’s used to identify proteins. The process is the same except we transfer the samples from the gel to the membrane via electroblotting instead of upward capillary transfer.

We use antibodies as probes to identify proteins of interest.

Question 30

Describe the process of electroblotting?

Answer 30

Used to transfer protein samples from gel to membrane.

Uses a nitrocellulose (PVDF) to capture proteins as they move from the cathode to the anode.

Once we capture the proteins we block the membrane with either BSA ( bovine serum albumin) or fat free cows milk. We then add the antibodies as probes which binds to the protein’s antigen.

Question 31

What are labels we can use to identify proteins?

Answer 31

They can be an enzyme of fluorophore.

We attach the label to a primary antibody or to a secondary antibody which binds to the primary antibody.

Question 32

Size- exclusion chromatography

Answer 32

Separation technique in which the stationary phase has small beads that captures small molecules while larger molecules elute through.

Question 33

Ion- exchange chromatography. What are the two forms?

Answer 33

Separation technique based on the stationary phase being coated with beads coated with an ion. It attaches the molecule of the opposite charge.
A salt is added to de- attach the molecule from the stationary phase.

Cation- exchange chromatography - The stationary phase is coated with anions which attracts cations.

Anionic- exchange chromatography - The stationary phase is coated in cations which binds anions.
- If PI is less than pH it’s negatively charged.
- If Pi is greater than pH it’s positively charged.

Question 34

What effects does changing the pH have on charge on protein?

Answer 34

• If protein is positively charged raising the pH makes it less positive as its side chains are deprotonated.
• If proteins is negatively charged and you decrease the pH it becomes less negative as it’s protonated.

Question 35

What are tags in affinity chromatography?

Answer 35

A way to prevent having to create a separate column for each protein we want to separate. The beads that bind the tags are sold commercially.

myc- tagged proteins are eluted once you add the myc sequence.

Polyhistidine tag - an imidazole ring that is eluted once you add nickel.

Question 36

Describe co- immunoprecipitation?

Answer 36

A variation of affinity chromatography in which we examine binding interactions.

Beads with an antibody is at the bottom of a test tube. The protein mixture is allowed to mix and the protein of interest binds to the antibodies. Proteins that are bonded to the proteins of interest (those proteins bonded to the beads) is said to be co- immunoprecipitated (they fall out of bulk solution).

We remove the supernatant (overlaying solution) and use a wash to remove any impurities.

Use western blot to separate the protein of interest and the co- immunoprecipitated protein.

Question 37

What’s the point of performing dialysis?

Answer 37

Samples for analysis can have contaminates or a pH or salt concentration not compatible with later experiments.

Question 38

Describe the process of dialysis?

Answer 38

Sample is placed in container with a selective membrane that allows salt, water, and impurities through but not desired products.

Water and undesired materials flow out across the membrane into the dialysate. Desired products diffuse out of dialysate into the membrane.

osmotic pressure is equalized across the membrane due to the diffusion of solutes until they reach equal concentrations across membrane.

Question 39

Hemodialysis

Answer 39

Dialysis performed in humans to perform function of kidneys.

Question 40

What is quantification?

Answer 40

Measuring the amount of protein in a sample after collection of the protein.

Question 41

Absolute quantification

Answer 41

A way to quantify the number of proteins in a sample by measuring the amount in terms of molarity or mass per volume.

Technique must be performed prior to separation to make sure changes is due to expression and not loading errors.

Question 42

UV- Vis Spectroscopy

Answer 42

Quantifies the amount of protein by measuring the maximum wavelength it can absorb (lambda max).

Amount of protein is directly proportional to the absorbance of visible or UV light.

Best used for pure samples as impurities can distort the UV- VIS absorbed.

Question 43

How can we separate proteins from impure samples?

Answer 43

First we create a standard which is a curve graphed at absorbance v. concentration of a protein in a sample of known concentration.

We add a stain to the protein of interest which creates an intense visible color that we can graph.

If the concentration of protein of interest goes over what the instrument can handle, we can perform dilutions.
From the dilution we can determine the original amount by multiple amounts by the dilution factor.
If serial dilutions are performed, we can multiply by dilution factors many times.

Question 43

How to get the absorption coefficient?

Answer 43

Either get it from the literature value or from estimating it by using the number of tryptophan residues.

Question 44

Binding assay

Answer 44

A way to measure the interaction between a protein and it’s ligand.

We measure via the Kd

The bound protein + ligand has different absorbance than free protein and ligand.

Question 45

Isothermal titration calorimetry

Answer 45

A way to measure ligand + protein interactions by looking at the change in enthalpy.

A vessel is at constant temperature and has purified proteins, ligand is gradually added, and the enthalpy is measured.

If reaction is exothermic, the power needed to maintain the temperature is decreased. This change is plotted against the baseline (the power needed to maintain the temperature).

We can calculate enthalpy by calculating the area under the peak formed from baseline and peak.

Technique doesn’t require free ligand approximation.

Question 46

Tm and the DSC (Differential scanning capacity)

Answer 46

Tm (melting temperature) is the temperature in which 50% of nucleic acids is denatured and 50% is hybridized.

As the nucleic acids are denatured the heat capacity increases, we can measure it by using the differential scanning capacity.

Question 47

Describe the process of differential scanning capacity (DSC)

Answer 47

Used to measure the Tm.

DSC increases the temperature at a constant rate while monitoring the power needed to maintain that rate.

As samples denature the power increases while the temperature decreases (breaking bonds = endothermic process) because more and more bonds are broken.

The Tm is the highest peal and the point of highest power output. After the Tm the power needed decreases because fewer bonds are left.

Question 48

How can we measure the Tm with fluorescence?

Answer 48

We use a fluorescence that binds to native protein or dsDNA, dsRNA. We measure the decrease in the signal as they denature.

To measure the Tm we measure changes in protein conformation. It takes on a sigmoidal shape (because of positive cooperativity). 50% denatured is the Tm.

Question 49

Describe circular dichroism

Answer 49

Used to measure the secondary structure of proteins.

We measure secondary structure by the degree a secondary structure absorbs right- handed or left- handed directions of circularly polarized light.

The two secondary structures absorb the right-handed or left-handed at different extents. This is referred to as the ellipticity.

Question 50

Cryogenic electron microscopy

Answer 50

A way to determine the structure of a protein by freezing them and using electron microscopy to take 2D images of them called projections. We take multiple projections at different angles to create a 3D image.

Question 51

X- Crystallography

Answer 51

When a protein sample is purified and concentration until crystals appear. X- rays are shined into structure which interacts constructively or destructively. We get the image from this.