Native electrophoresis
Speed and direction (migration) depends on protein's net charge, uncharged proteins stay still
SDS-PAGE electrophoresis
Speed and direction (migration) depends on mass onlyUses amphipathic detergent called dodecyl sulphate (DS): unfolds proteins into linear chain and sulphate applies negative net charge w m/z ratio to all proteins uniformly
DS
dodecyl sulfateamphipathic detergent
reducing conditions in gel electrophoresis
imitates cytoplasmbreak disulphide bridges of Lys residues via addition of reducing agents like beta-mercaptoethanol
what tracks success of purification steps
electrophoresis + purification table
Specific activity EQN
spec. activity = (activity)/total mg proteinHIGH when (high activity)/(low mg total)LOW when (high activity)/(high mg total): impurities have not been removed yet, mg decreases as it does
Yield
activity now/activity at start
Purification factor
spec activity now/spec activity at start
Why does enzyme activity decrease?
When some arbitrary enzyme units get lost from assay used for testing
fractionating based off of:1. density and solubility2. size3. charge4. specific binding
1. centrifugation and precipitation2. size-exclusion chromatography and dialysis3. ion-exchange chromatography4. affinity chromatography
By centrifugation
Sedimentation of particles depending on mass, volume, and shape supernatant is floating at top, pellet is at bottom
By precipitation
Salt out using ammonium sulphate: high [salt] enhances hydrophobic effect and encourages protein precipitation Increase [AS] in stages w centrifugation after each precipitation stage
Salting out
high [salt] enhances hydrophobic effect because of hydrophobic patches on protein surface, water entropy increases and apolar groups stick closer
By dialysis
After collecting precipitated protein dialysis will remove AS salt Via dialysis bag in which small salt molecules diffuse out of permeable bag, larger molecules stay inside
Stationary phase
beads made out of resin packed in tubular column
Mobile phase
buffer that contains proteins past the stationary phasetransient interactions with the stationary phase can be based on ion charge, binding, etc. which slow down elution down the column
ion exchange chromatography
buffer contains soluble proteinse.g., cation-exchange resin in which beads have neg chargesome proteins will bind tightly (strong pos charge), some proteins will bind weakly (low pos charge): elution occurs in order of increasing charge bc higher charges will be binded for longer/strongerelute by adding salt to weaken ionic interaction bw protein and resin
cation/anion exchange chromatography
resin can either be cation-exchange (resin has neg charge bc protein has pos charge and is a cation) or anion-exchange (resin has pos charge bc protein has neg charge/is an anion)aka name correlates w protein character
size-exclusion chromatography vs. gel electrophoresis
size-exclusion: proteins carried in mobile phase, delayed because they get diffused away into porous beads, smaller proteins diffuse more so larger proteins elute fasterelectrophoresis: proteins driven through gel, delayed because of gel friction, smaller proteins travel faster so larger proteins elute slower
specificity in affinity chromatograph
only right protein to bind to, not only similar proteinsto the extent that if you bind your desired protein to a columned can wash off all other proteins; to elute yours you add a molecule that will compete with resin group to bind to your protein
desired protein and elution for following groups attached to resin:1. DNA w recognition sequence2. antigen3. Ni 2+4. antibody for known epitope/peptide
1. TF, elute w free DNA2. antibody, elute w free antigen3. protein w His tag, elute w imidazole bc it binds w Ni2+4. protein w epitope tag, elute w free epitope - can elute w high salt too for all of them - for tags: have to add tag sequence to protein sequence via recombinant DNA first
Variable V domain
Has hyper variable loops (CDR) unique to its antibody and complementary to one epitope
Constant C domain
Same in all antibodies, not unique
Fc: physiological role
doesn't interact with antigen BUT host cells do bind to it to initiate antigen destruction (link fluroscent dye on it during immunofluroescence microscopy)
Epitope
Region on antigen that is recognized by antibodyEach antibody has two V domains (one on each Fab fragment) that targets epitope complementary to that antibody
monoclonal antibodies
from one clonal/genetically uniform line of beta-lymphocytes that recognize one epitope on one antigen
polyclonal mixture of antibodies
mixture of multiclonal antibodies all against one antigen that has multiple diff epitopes
How does co-immunoprecipitation aid if I have an antibody attacking my protein ?!?
it can DISCOVER other proteins that bind to my protein
(Co)Immunoprecipitation Workflow
1. attach antibody to resin beads (like done in affinity chromatography) 2. immunoprecipitation: put beads in protein mixture/buffer for centrifugation to isolate bead-antibody and bound antigen 3. co-immunoprecipitation: if antigen is in complex with other proteins, isolate this again
affinity chromatography
synthesize resins w key functional groups to get interactions w more specificity
How does immunofluorescence aid if I have an antibody attacking my protein ?!?
can locate your protein/antigen in a cell
immunofluorescence: how to
covalently links fluorescent dye to Fc fragment of attacking antibodyallows us to visualize diff antigens by using differently dyed antibodiesidentify which gel band is ur protein in electrophoresis via immunoblot/western blot
immunoblot/western blot: workflow
identifies which gel band is ur protein in electrophoresis1. transfers unknown gel bands/blots from the gel to plastic membrane2. puts known antibody w dye (linked during immunofluroescne microscopy) on the membrane and wash off wtv doesnt stick3. find where antibody stuck to membrane and match location on original gel!
Reporter enzyme
converts invisible substrate into visible product e.g., peroxidase makes black producte.g., b-galactosidase from lacZ
ELISA
Enzyme-linked ImmunoSorbent Assayquantifies how much of your protein/antigen is their
ELISA workflow
1. isolate antigen from antibody2. link antibody with reporter enzyme3. immobilize/adsorb unknown antigens on test tube surface4. apply enzyme-antibody and wash off whatever doesn't stick5. apply substrate which will produce color, color intensity is a measure of antigen
Polyclonal antibodies and bright signals
two methods 1. use polyclonal mixture that is all enzyme linked, will all bind to different epitopes of your antigen2. layering strategy: primary polyclonal antibodies first bind to your antigen, then secondary enzyme-linked antibodies bind to invariant fc region of primary polyclonal antibodies - note: anti fc antibody has to be grown in diff host than fc used as antigen