RPC, HIC, Affinity Chromatography

Initial conditions of the mobile phase in RPC

polar

both the sp and proteins hydrophobic patches are hydrated with ordered water

elution strategy in RPC

[non polar solvent] gradient

gradually increase [non polar solvent] i.e. hydrophobic groups are those with long hydrocarbon chains

weakens hydrophobic effect affection elution

elution trend in RPC

less hydrophobic proteins elute 1st

more hydrophobic proteins require higher [non polar solvent]

HIC basis for elution (how are proteins affected)

proteins are loaded at a very high ionic solution (e.g high salt) which strengthens hydrophobic interactions

eluted by gradually reducing salt

salt acts as a dial

affinity chromatography exploits…

specific interactiosn between a protein and a ligand

affinity chromatography allows purification of a target protein from complex mixtures based on…

binding specificity

immunoaffinity utilizes

antibodies that are specific to your protein

antigen binding sites bind tighly to their target

procedure in immunoaffinity

antibodies are attached to the SP

elution requires certain conditions like low pH to disrupt strong antibody protein interactions

immunoaffinity is beneficial for high __

specificity

metal chelate affinity chromatography prerequisite

protein must be expressed recombinantly with an affinity tag

what is an His-tag

strong of 6 histitidines added to n or c terminus

binding of support of his-tag in stationary phase (beads)

his tag binds strongly to a bead that has a Ni2+

elution in metal chelate affinity

a competing imidazole is gradually added

as [imizadole'] increases it competes with his tag for binding to nickel —> displaces target protein

dialysis as a seperation method

purification buffer exchange method: can repleace undesirbale mobile phase with a protein friendly buffer

semipermable membrane (dialysis bag) to seperate molecule based on size

pores of pag are tuned to allow molecules below a certain size to pass through while retaining larger molecules

describe precipitation of proteins

seperates a protein from a solution as a solid by altering the solubility with the addition of a reagent

key principle of precipitation

soluable state: protiens are disperesed as individual particles in solution

precipitation: envriomental changes trigger molecule to aggregate

describe salting in

low salt concentrations will increase solubility

(salt will shield the charges that cause proteins to stick together preventing aggregation)

describe salting out

as aalt concentration increases it competes for water which removes water from protein surface

with less water to interact with more likely for them to aggregate effectively “drying out” the proteins