Lecture 3 - Studying Proteins

Question 1

What are the steps of SDS polyacrylamide-gel electrophoresis?

Answer 1

1) Proteins are boiled in SDS detergent to denature them and make them negatively charged
2) Electrical current forces passage through polyacrylamide gel towards the positive pole
* Smaller proteins migrate faster through pores of the gel

Question 2

What are the ways proteins can be detected after SDS-PAGE?

Answer 2

1) Protein stain - e.g., coomassie blue
2) Western blotting - Gel is transferred to a membrane and gets probed with an antibody against the protein that recognizes a specific known protein of interest
3) Mass spectrometry - identities unknown protein

Question 3

What are sources of protein for studying their function?

Answer 3

Purified from the organism/cells by:
1) Cell fractionation combined with in vitro assay (old way)
2) Immunoprecipitation (need antibody against protein)
3) Express a tagged version of the gene in E. coli (or invitro) or in animal cells (easier to purify the protein from bacterial cells)

Question 4

What method is immunoprecipitation?

Answer 4

Rapid affinity purification method

Question 5

What are the steps of immunoprecipitation?

Answer 5

1) Grind up cells from a mixture of proteins extracted from the cell in extraction buffer and centrifuge to obtain total cell extract
2) Add an antibody against a specific protein
3) Couple the antibody with something heavy (e.g., agarose bead that will naturally fall to the bottom of the tube)
4) Centrifuge to separate soluble protein of interest from insoluble proteins (keep pellet)
5) Elute the protein by adding something to break the tight bond between the antibody and protein

Question 6

How to purify a protein if there is no antibody against it?

Answer 6

1) Clone the gene into a plasmid (expression vector)
2) Express a gene and allow it to be transcribed (cut DNA with a restriction endonuclease, insert the protein-coding DNA sequence, and introduce recombinant DNA into cells)
3) Translate overexpressed mRNA into overexpressed proteins

Question 7

How is purified protein obtained after expression in E. coli?

Answer 7

1) Clone the gene into a vector that contains an epitope or affinity tag
2) Express the fusion protein in bacteria (or in vitro)
3) Purify the fusion protein by affinity chromatography or immunoprecipitation

Question 8

What is an epitope tag?

Answer 8

Small protein sequence (10-12 aa) that is recognized by well-characterized and commercially available antibodies (e.g., HA tag)

Question 9

What is an affinity tag?

Answer 9

Protein that can be easily purified based on affinity to a specific substrate? (e.g., GST tag - has an affinity to glutathione)

Question 10

What can tagged proteins in bacteria be used for?

Answer 10

Used in in vitro assays, to test for direct interactions between two proteins by GST pulldown, determine protein structure, and for antibody production (structure protein localization and perform co-immunoprecipitations to identify interacting proteins)

Question 11

How is a protein fusion made?

Answer 11

1) PCR amplify your gene from cDNA
2) Clone gene into a vector that already has an epitope or affinity tag in it
* Fusing two protein sequences, not using two proteins together

Question 12

How is PCR used to insert an epitope tag?

Answer 12

• PCR primers have restriction enzyme sites at their ends which can clone PCR product into a plasmid vector
• One of the primers also has a sequence for an epitope tag (as long as the primer has enough of a complementary sequence, it will recognize the template and PCR)
• Alterations can be made that don’t match on the 5’ end that encodes something
• PCR product will be the full altered sequence with restriction sites on each side

Question 13

How is GST affinity purification carried out?

Answer 13

1) Clone gene into GST vector (the expression vector with GST needs to get the coding sequence of the gene into the vector by using the 5’ and 3’ primers to cut restriction sites and ligase to seal the nicks)
2) Express GST-protein
3) Bind GST-protein to glutathione (GSH) beads
4) Wash beads and elute protein with glutathione since the extra free glutathione will compete with the beads
5) Run eluate on protein gel

Question 14

What is GST pulldown and how is it done?

Answer 14

Tests for in vitro interaction between two known (and cloned) proteins
1) Bind GST-protein to glutathione beads
2) Incubate with another protein
3) Wash beads and elute with glutathione
4) Run eluate on gel
* If protein stays with beads after elution, it will be stuck to the protein and be pulled down

Question 15

How do you express protein in animal cells?

Answer 15

A tagged form of protein in cultured cells or in a whole organism can be expressed because the vector contains a strong inducible promotor
- Transfect into animal cells or transform into animals to obtain transgenic animals
- As with bacterial expression, the gene is cloned with an epitope or affinity tag (or with GFP)
- Purify protein with epitope or affinity tag
- Directly study protein localization in animals cells
- Directly perform co-immunoprecipitation (or affinity purification) to identify interacting proteins

Question 16

What is co-immunoprecipitation and what are the steps?

Answer 16

Way to purify proteins and identifies interacting partners
1) Grind up cells in mild extraction buffer to preserve protein-protein interaction and centrifuge to obtain total cell extract
2) Add antibody against specific protein
3) Add beads coupled to protein
4) Centrifuge and keep the pellet which contains the beads attached to the antibody-specific protein
Proteins that are associated with the immunoprecipitated protein will co-immunoprecipitate with it - can be identified by Western blot or mass spectrometry

Question 17

How to detect specific proteins?

Answer 17

Western blot and probing with antibodies

Question 18

How to perform a Western blot?

Answer 18

1) Run proteins on gel and transfer to membrane
2) Probe with antibody against the protein to determine interactions

Question 19

How can unknown proteins be identified after co-IP?

Answer 19

1) Run eluate on SDS-PAGE
2) Stain gel
3) Cut out prominent beads
4) Mass spectrometry to identify proteins

Question 20

What is mass spectrometry?

Answer 20

A highly sensitive method for identifying unknown proteins by using specific mass to charge rations from a peptide that can only be achieved by a certain combination of amino acids since measurements are so precise

Question 21

What are the steps of mass spectrometry?

Answer 21

1) Take complex mixture and break them into smaller peptides, typically by adding enzymes
2) Ionize peptides
3) Put ionized peptides into a machine that will separate them according to mass
4) Read mass to charge ratio for each peptide

Question 22

What is a BLASTp search?

Answer 22

It identifies related proteins in the same or differing species, identifies homologs, paralogues, and similar proteins, and can get an idea of the relationship between your unknown protein and others

Question 23

What can domain prediction identify?

Answer 23

Putative domains (looks like domain we’ve seen before but don’t know if it’s functional) based on sequence similarity to characterized domains

Question 24

How can protein structure be determined using x-ray diffraction/crystallography?

Answer 24

Protein is expressed in E. coli and affinity purification is done to get a highly pure soluble protein so that it can form crystals by dehydrating it - Protein crystals are then subjected to x-ray (pattern diffraction used to determine protein’s crystal structure)

Question 25

What is an alternative to x-ray crystallography?

Answer 25

NMR - does not require crystalization so you don’t need a purely soluble protein; not as precise; does not work for large proteins

Question 26

Why is immunofluorescence used to detect specific proteins in cells?

Answer 26

Used because there are size limits to microscopes - Living cells can be seen clearly in a phase-contrast or a differential-interference-contrast microscope, but not under light microscopy unless the proteins are labeled and fluoresce

Question 27

How is immunofluorescence used to detect specific proteins in cells?

Answer 27

• An antibody is coupled to something that fluoresces (e.g., antibody that recognizes tubulin can be coupled with something that fluoresces green)
• Different colours can be used to fluoresce different areas of the proteins in a cell

Question 28

How are antibodies generated?

Answer 28

1) Purified protein is obtained from bacterial expression, in vitro translation
2) Inject into rabbit, mouse, or other animal
3) Obtain serum that contains antibodies against multiple epitopes on protein and non-specific antibodies after several weeks
4) Serum is put into a tube where the protein of interest is attached to beads and the antibody will bind to the protein while the others get washed away
5) Specific antibodies can be purified by affinity purification

Question 29

How are monoclonal antibodies generally obtained in mice?

Answer 29

1) Inject protein and let it amount an immune response
2) Remove spleen
3) Harvest antibody-secreting B-cells (each B-cell secretes an antibody that recognizes a single part of the protein)
4) Fuse B-cells to myeloma cells - hybridoma cells can divide indefinitely and produce a single antibody
5) From multiple hybridoma cell lines, identify one that makes an antibody specific to the injected protein

Question 30

How can antibodies be used to detect specific molecules?

Answer 30

1) Fix cells onto the slide (e.g., by formaldehyde and cross-links to make it stable)
2) Add antibody against the antigen and incubate so that it finds the protein
3) Detect the protein with a secondary marker-coupled antibody that binds to the primary antibody and amplifies the signal to some degree

Question 31

How can antibodies raised in different species be used to detect specific molecules?

Answer 31

1) Add both antibodies so they find their target antigens
2) Add both tagged secondary antibodies against the primary antibodies that are from different animals so they find their target protein
3) Cells fluoresce to indicate where both proteins are and their relation in the cell can be studied

Question 32

What are some uses of antibodies?

Answer 32

• Identify protein in Western blots
• Immunofluorescence
• Use for immunoprecipitation

Question 33

What are some alternatives to antibodies?

Answer 33

Clone into a plasmid for transforming into animal cells by expressing as a fusion protein in vivo
- Epitope tag - Western, IF
- Individual proteins can be fluorescently tagged in living cells and organisms using GFP by cloning a gene into a plasmid that has a GFP sequence and a strong promoter for animal cells, transforming into living cells, and imaging them with a light microscope

Question 34

What are some pros to bacterial cell expression

Answer 34

• Allows for purification of larger amounts
• Generally more pure which makes it better for determining the crystal structure
• Better for antibody production
• Often better for in vitro studies

Question 35

What are some pros to animal cell expression?

Answer 35

• Can directly study protein localization and interacting partners
• May be better for in vitro studies because protein is processed properly (glycosylation, phosphorylation, etc.)