Midterm No. 1 Lab Techniques

Question 1

What does an SDS-PAGE / 1D gel tell us?

Answer 1

Shows the approximate molecular weight of a protein of interest

Question 2

Steps to an SDS-PAGE / 1D gel

Answer 2

1. SDS ionic deterget is used to make the proteins negatively charged over all surface area
2. A reducing agent (mercapethanol) adds hydrogen to the proteins to break disulfide bonds and other interactions
3. Proteins are heated to further denature
4. Samples rune on the PAGE, a porous jelly gel. A ladder with known weights and size is run parallel to contextualize the samples

Question 3

What is SDS and what is it used for?

Answer 3

It’s the ionic detergent. It makes proteins negatively charged over their entire surface area

Question 4

What is the purpose of the reducing agent in the SDS-PAGE 1D gel?

Answer 4

It adds hydrogens to the proteins to break disulfide bonds and other interactions

Question 5

What experimental questions (aside from determining approx. molecular weight!) can an SDS-PAGE 1D gel be used for?

Answer 5

How long until the protein of interest is expressed in cell extracts after I introduced it?

Did the protein get heavier or lighter after modifications?

Has the protein been degraded?

Question 6

What is an Isoelectric Focusing 1D gel used to tell us?

Answer 6

Tells us the overall charge of a protein from its collection of amino acids, whether its basic or acidic

Question 7

What charge do proteins have at low pH?

Answer 7

Positive

Because they tend to be negatively charged (they still have a unique charge, but most tend towards negative), they snatch up the excessive positive charges (H+) from the environment

Question 8

What charge do proteins have at high pH?

Answer 8

Negative

Proteins tend to usually be negatively charged. At high pH they will still be negatively charged because there’s not enough H+ to go around

Question 9

Steps to an Isoelectric Focusing 1D test

Answer 9

1. Hook a pH gel to an electric field. This will force protein movement to their isoelectric points
2. Load samples and run the gel
3. Based on the proteins isoelectric points, we can determine their overall charge and whether they are acidic or basic

Question 10

What test should you run to determine wheter a protein has become phosphorylated?

Answer 10

Isoelectric focusing 1D gel

Also possibly a Western Blot + 2D gel

Phosphorylation increases negative charge, affecting acidity

Question 11

Steps to an SDS + Isoelectric 2D gel

Answer 11

1. Use an isoelectric pH gel strip to separate a protein mixture by charge
2. Soak the gel strip with SDS and a reducing agent. This strip will now act as wells for the electrophoresis
3. This strip will now act as wells for the electrophoresis. Run the gel to separate proteins by weight

Question 12

What can Western Blots NOT be used for?

Answer 12

To directly discover a never before seen protein

This is because Western Blots run off of antibody recognition, and in order to make the antibodies specific we need to already know the target protein

Question 13

What is the job of the primary antibody in a Western Blot?

Answer 13

To bind to a specific protein of interest

Question 14

What is the job of the secondary antibody in a Western Blot?

Answer 14

To bind to the primary antibody. It’s equipped with a synthetic fluorescent marker or some other visual tag

Question 15

What questions does a Western Blot + SDS PAGE gel answer?

Answer 15

The antibodies from the Blot and the gel from the SDS PAGE gel allow us to see if our target protein is present in our sample mix

Also used to determine concentration levels. Dark bands indicate high levels, light bands indicate low levels

Question 16

What questions can a Western Blot + 2D gel answer?

Answer 16

Did my specific protein get phosphorylated?

Is my specific protein present after a certain mutation?

Question 17

What is immunofluorescence + colocalization used for?

Answer 17

Immunofluorescence uses fluorescently tagged antibodies to bind to proteins within a living cell. The localizations of the antibody+proteins can be seen under a microscope

Using two different fluorescence colors on two different antibodies allow us to see points of overlap, aka colocalization

Question 18

What is immunoprecipitation used for?

Answer 18

To extract proteins from a mix

Question 19

Steps to immunoprecipitation

Answer 19

1. Antibodies specific to a protein of interest are added to a mix
2. Non-bound proteins are removed
3. Antibodies are removed

Question 20

What is Co-Immunoprecipitation used for?

Answer 20

To show if two proteins actually interact

Does my target antigen bind to or associate with other proteins? Is it in a complex with other proteins?

Question 21

Steps to Co-Immunoprecipitation

Answer 21

1. Use an antibody for a known protein
2. The antibody-bound protein interacts with other unknown proteins in a larger complex
3. Chemical solvents or antibody breads help remove the anitobdy and whatever is attached to it from the protein mix
4. Unknown proteins + known proteins are precipitated out
5. Bonus: other gel techniques can then be used to learn more about the unknown proteins

Question 22

Polyclonal antibodies

Answer 22

Several different varieties of antibodies that bind to one antigen

Made in animals when more than one B cell responds to a particular antigen

Question 23

Monoclonal antibodies

Answer 23

Single antibody species

Harvested from myeloma cancer cultures

Question 24

Pros of polyclonal antibodies

Answer 24

Cheap

Creates more than one variety of antibody for the same antigen

The different varieties may bind to different areas of the target antigen

The antibodies, due to their diversity, are more tolerant of small changes in protein structure, heat, shock, etc to the antigen

Question 25

Cons of polyclonal antibodies

Answer 25

Finite supply (the animal making them will eventually die)

Question 26

Pros to monoclonal antibodies

Answer 26

Infinite supply Single antibody species, highly specific

Question 27

Cons to monoclonal antibodies

Answer 27

Not cheap to make Will bind to only one specific site on the antigen Antibody isn't tolerant of small changes in the target antigen

Question 28

Why are co-immunoprecipitation experiments better than co-immunolocalization for showing if two proteins actually interact?

Answer 28

Co-immunolocalization only shows if two proteins are in the same area Co-immunoprecipitation shows if they actually interact

Question 29

Steps to X-Ray crystallography

Answer 29

1. Obtain a protein of interest 2. Purify the protein using fast protein liquid chromatography 3. Crystallize the protein using a vapor diffusion technique. All the molecules will be in the same orientation, thus forming a crystal and not an aggregate 4. Collect data from an x-ray diffraction experiment. The crystal must be super cold while subjected to the x-rays 5. Structure determination. The X-ray diffraction pattern is analyzed to yield an electron density map. The alignment of the electron densities are compared to the known primary sequence, and the protein model is generated

Question 30

How do researchers determine the structures of proteins and protein complexes?

Answer 30

X-ray crystallography Cryoelectron microscopy NMR

Question 31

What does PAGE stand for?

Answer 31

Polyacrylamide gel electrophoresis

Question 32

Steps to cryoelectron microscopy

Answer 32

Start with purified protein/protein complex in solution Dilute the purified sample Disperse the diluted solution onto an electron microscopy grid Freeze the grid with liquid helium and allow shapes to form Determine structure by observing the shapes from their different angles, relative to the known primary sequence

Question 33

Compare x-ray crystallography to cryoelectron microscopy

Answer 33

In both x-ray crystallography and cryoelectron microscopy, the protein of interest’s primary sequence must be known beforehand in order to interpret any meaningful results. The electron density map and shapes on the EM grid aren’t very useful on their own. They need to be contextualized with the known primary sequence.

Question 34

You're trying to run an SDS PAGE gel. What would happen if you forgot the SDS?

Answer 34

The SDS is the ionic detergent. Without it the proteins wouldn’t be coated with the negative charge and wouldn’t be kept apart, resulting in one large glob of proteins in the gel matrix because they couldn’t be separated by their molecular weight

Question 35

You're trying to run an SDS PAGE gel. What would happen if you forgot the beta mercaptoethanol?

Answer 35

The disulfide bonds stabilizing the proteins’ tertiary and quaternary structures wouldn’t be broken, resulting in an inaccurate sorting of proteins in the gel matrix as the proteins’ true molecular weights wouldn’t be clear (quaternary structures still together would make all proteins involved seem heavier than they are)

Question 36

You're trying to run an SDS PAGE gel. What would happen if you forgot to heat the samples to 95 C?

Answer 36

The proteins would not denature/unfold, and wouldn’t be able to properly sort themselves through the gel matrix

Question 37

You're trying to run an SDS PAGE gel. What would happen if you forgot to run a lane of molecular weight standards?

Answer 37

You’d get accurate results from your experimental lanes, but you wouldn’t be able to interpret or contextualize your results. Your experiment would be useless

Question 38

Where does the positive electrode go in a gel?

Answer 38

For the gel to work, the positive electrode will need to go at the bottom of the gel so that the negatively charged proteins will be attracted to it and run through the gel. If the positive electrode was at the top of the gel, the negatively charged proteins would run towards the top and out of the lanes, ruining the experiment.

Question 39

Steps to 2D PAGE gel

Answer 39

Separate proteins by their native charge using isoelectric focusing Individual proteins will move across a charged pH gradient to their electrically neutral state, aka their isoelectric point. Different proteins have different isoelectric points, are neutral at different pHs Incubate a tube gel with an existing stable pH gradient in the protein solution/cell lysate. This forms the first dimension Align the isoelectrically separated tube with the negative end of an SDS PAGE gel to form the second dimension

Question 40

Does it matter which method is applied first in 2D PAGE gel tests?

Answer 40

The end result is a 2D separation, in which proteins are separated by both their native charge AND their molecular weight. The order in which these tests are run is vital, as the SDS PAGE gel only works with the tube gel’s input, while the tube gel cannot work with the SDS PAGE gel input

Question 41

Western Blot steps

Answer 41

Load and separate protein samples onto an SDS PAGE gel Electrophoretically transfer the subsequent fractionated proteins onto a PVDF membrane Block the membrane with a neutral protein, usually milk/casein powder Incubate the membrane with the primary antibody specific to your target protein Incubate the membrane with an HRP-labeled secondary antibody specific to the primary antibody Incubate the blot with chemiluminescence

Question 42

Steps to a DNA footprint assay

Answer 42

Cloned gene fragment is amplified and is radioactively labeled at one end, then incubated in samples with and without its cell extract (RTFs) The genes are then chopped with DNase after incubation. The sample cell without the cell extract is chopped the full gradient length, while the sample with the cell extract is missing a section of the chop gradient Fragments are run on a gel The gel is layered with x-ray film so that only the radioactive fragments are shown The missing chunk in the sample with cell extract shows a footprint where the RTF binds, as the DNase was blocked by the RTF

Question 43

Steps to a gel shift assay

Answer 43

Tool used to identify RTFs and the DNA elements they bind to A DNA fragment of interest is fractioned by some metric (weight, charge, etc), then mixed with RTF/cell extracts The DNA is then bound to a radioactive probe The samples are run on a gel, then overlaid with an x-ray film so that only the radioactive samples are shown DNA bound to a RTF protein won’t travel as far as free DNA. The x-ray film looks for free probes vs bound probes

Question 44

How is binding strength determined?

Answer 44

Use stress tests. Ex: increase thermal energy to make it harder for proteins to bind Ex: decrease concentration of reactants to decrease the ratio of AB dimers`

Question 45

How do we know which cyclin types are present at which times and phases of the cell cycle?

Answer 45

Work with Western Blots

Question 46

How did we learn about the different levels of DNA packaging?

Answer 46

We learned about histones and nucleosomes and their “beads on a string” characteristics by using transmission electron microscopy We learned how long the strands wrapped around each nucleosome are through nuclease digestion. The nucleases chewed up the DNA but left the histones intact, and when treated with salt the DNA was released and the average number of base pairs per octamer was revealed (it’s ~147, with ~50 bps making up the linker strand, so a total of ~200 bp per nucleosome)

Question 47

What is RNAseq and how is it used?

Answer 47

Comparison of gene expression in different cells or in different conditions Asks how cells can change their patterns of gene expression in response to changed conditions Reverse transcriptase transcribes mRNA molecules into DNA, the DNA is then sequenced This technique shows both wha’ts being transcribed and relative transcription levels, i.e. how much mRNA is being produced from a single gene This data is graphed via Log2 FoldChange