MBB lecture 14-15

First step for protein purification

break open cells by sonication, shearing, and/or treatment with mild detergents

what is protein purification a first step for?

determining proteins structure and funtion

4 ways to break open cells (for purification)

1. sound waves pop them open

2. mild detergent makes holes on plasma membrane

3. squish chamber with piston and push cells through pore until they pop

4. squish cells between rotating plunger and glass vessel

how does centrifugation separate macromolecules

density (often an initial step)

what order are things moved in differential centrifugation

nuclear fraction, mitochondrial fraction, membrane fraction (then you just have cytosol fraction)

salting out

proteins=less soluble in high salt so will precipitate out

(different proteins at different salt conc.)

happen at pH where protein is neutral (net charge 0)

when are proteins least soluble

at neutral (net charge 0)

column chromatography

separates based on differential physical or chemical interactions with a solid gel matrix

types of column chromatography

1. ion exchange

2. size exclusion (gel)

3. affinity

ion-exchange chromatography

separates based on charge (anion exchange: protein is anionic the column is positive)

in anion exchange positive leaves first= is repelled

gel chromatography (size exclusion)

separates based on size (biggest come out first because they dont have to go in tubes)

Affinity chromatography

separates based on specific ligand interactions

(target protein interacts with ligand covalently bound to resin, non specific proteins flow through column without interacting)

SDS-PAGE: PolyAcrylamide Gel Electrophoresis

separates based on mass

preformed on vertical apparatus, buffers in top and bottom chambers, 2 chambers connected by gel matrix between 2 glass plates, sample added to top with pipette, external power source allows current to flow through, migration of negatively charged proteins toward anode (positive) side of chamber

Estimation of protein MW in SDS-PAGE: use MW markers

The electrophoretic mobility of proteins in SDS-polyacrylamide gels is inversely proportional to the log of their MW

what is a dye front

leading edge of dye migration during electrophoresis Rf: retention factor

Rf=

distance migrated by protein divided by distance migrated by the dye front

protein ladder

mixture of proteins of known molecular weights that run alongside unknown protein samples during electrophoresis process. A reference to estimate molecular weights

2D PAGE

used to separate proteins based on both pl and MW.

separated in first dimension by isoelectric focusing (based on overall charge). then separated in 2nd dimension by standard SDS-PAGE

protein spots can be excised from gel and identifies using mass spec.

what is 2D DIGE

2-d differential in-gel electrophoresis

what dyes does 2d DIGE use and for what

Cy3 (green) and Cy5 (red) to distinguish 2 protein samples run on the same 2D PAGE gel

(proteins unique to one sample show one colour, proteins in both samples have a mixture colour)

what is proteomics

large-scale study of proteins (mostly structure and function)

does proteomics usually refer to the entire set of proteins for an organism

yes usually refers to that (proteome) or set of proteins present in particular cell type under certain conditions

does the proteome differ from cell to cell and change?

yes and constantly changes depending on interactions with environment

tools for proteomics

isoelectric focusing, protein arrays and chips, co-immunoprecipitation (co-IP), mass spec (MS)

approaches are, by necessity, “high-throughput” meaning lots of proteins studied/processed at the same time

the proteomics approach (steps) (7)

identifying all proteins in given sample

1. sample (could be for ex. lysate from organ, secreted proteins from bacterium, cytoplasmic contents, membrane fraction)

2. prep steps

3. isolate proteins (1D PAGE, 2D PAGE)

4. cut protein spots out of gel

5. digest (cut up) with protease (trypsin)

6. mass spec

7. protein id and informatics

NMR (6 points)

• nuclei align in electromagnetic (em) field according to spin

• nuclei can “flip” if given pulse of em radiation at “resonance frequency “

• energy absorbed to flip nuclei is recorded as line or peak on an NMR spectrum

• energy required for flip depends on surroundings of nuclei

• spectrum gives info about structure

• proteins need to be little (<50kDa)

X-ray crystallography (7 points)

• expose protein crystals to x-ray beam, collect x-ray diffraction data

• big things like viruses can use this method

• need to be able to crystallize protein (getting diffraction quality crystal is rate limiting step)

• crystals are grown in buffer containing “precipitant” that reduces solubility of protein and favours formation of ordered crystals

• in protein crystal all molecules are oriented the same way in a 3D lattice

• proteins not fully hydrated so not in native form/environment

• membrane proteins need detergents to solubilize them (hard to crystallize)

what are antibodies

• also called immunoglobins are proteins made by animals in large amounts response to presence of antigen

what are antigens made of

protein, polysaccarides, nucleic acids, small molecules

why are antibodies useful

• because of high specificity

• they bind to their antigen with high specificity and affinity

• can be used to identify proteins and their locations in the cell, to purify proteins, quantify them, and to identify protein binding partners

antibody structure

• made of 4 chains (2 heavy, 2 light)

• each heavy and light pair form 2 identical antigen-binding sites

• chains are held together by disulphide bonds and non-covalent interactions

what is the F(ab) domain (antibody) and what site on the antigen does it bind to

antigen-binding fragment, epitope

what is the F(c ) domain (antibody)

binds to receptors on host cells (crystallizable fragment)

why do antibodies bind to antigens

stereochemical complementarity between anitgen binding site and epitope on the antigen

antibody production (rabbit example) (8 steps)

1. immunize 4 times over 6 weeks

2. obtain blood sample

3. blood serum contains mixture of nonspecific and antigen specific antibodies

4. purify using affinity chromatography (load, wash, elute)

5. after load flow through fraction comes through

6. after wash nonspecific antibodies come through

7. after elute elution of antigen specific antibodies

8. end up with affinity-purified rabbit polyclonal antibodies

purpose and steps for Enzyma linked immunosorbent assay (ELISA) (7 steps)

• to quantify the protein antigen based on amount of primary Ab bound

1. antigen coated well

2. wash

3. specific antibody binds to antigen

4. wash

5. enzyme linked antibody binds to specific antibody

6. wash

7. substrate added and converted by enzyme into coloured product (rate of colour formation is proportional to amount of anitbody)

what is immunoblotting (western blots)

antibody based detection and quantification of protein antigen of interest.

protein has been electrophoresed on SDS-PAGE gel and transferred to membrane prior to incubation with primary antibody

steps of immunoblotting

what is immunoflourescence

an antibody-based technique used to identify proteins in cells that have been chemically treated in a way that preserves cell architecture