BCH-Week 4

Question 1

what are some features used to purify proteins

Answer 1

differences in size, sequence, charge, and presence of specific binding sites

Question 2

what is teh first step to do to be able to purify the protein of interest

Answer 2

the first step to be successful in isolating and purifying the protein of interest is to choose teh correct cell type and choosing teh right method for cell lysing which depends on how fragile the protein is

Question 2

why is protein purification important

Answer 2

to study the portein structure and function

Question 3

what are some methods use to break open the cell and obtain protein of interest

Answer 3

mechanical and physical methods such as grinding, sonication, or vortexing with glass beads but if these methods generate lot of heat, tehy could cause issues
osmotic pressure but it risks the diruption of target if too fragile since hypotonic solution will cause the cell to explode
chemical bases can be used
but all of the above methods have their own downsides like pH and temp ranges altering protein of interest

Question 4

what is a safe way for cell lysis that doesn’t deal with pH and temperature change

Answer 4

centrifugating

Question 5

describe the step involved in cell centrifugation

Answer 5

this process will allow for seperation of protein of interest safely and by preparing a crude extract that will be centrifuged to seperate teh supernatant from pellet which contains the largest molecule/organelle in solution
the centripital force is used to spin teh solution and seperate components based on their mass
teh nucleus is the first to get seperated since it is the biggest in mass, after removingit and centrifugate again, the mitochondria will be in the pellet as teh second one to get seperated
then the final seperation is of the microsome which leaves only proteins and metabolites in the supernatant

Question 6

what does the Beer Lambart law tells us

Answer 6

it is useful concept in spectroscopy as it relate the absorbance of light by substance to its concentration
it has teh formula of : A= e c l

Question 7

how can we use the Beer law to learn about teh protein’s structural component

Answer 7

the protein’s concetration can be measured using absorbance at 280nm by the aromatic rings present since most proteins are colourless and don’t absorb visible light

Question 8

what stains can we use to quantify concentration of protein or visualize the proteins

Answer 8

exampel will be coomassie blue stain which will help us visualize teh protein when hit with UV light and emits it to be quantified

Question 9

What is the general idea behind chromotography

Answer 9

the differential partioning of proteins by having a mobile phase, solution, and a stationary phase, the column, which will have characteristiucs to help us seperate teh protein of interest from other protein

Question 10

What are the different types of chromotographies

Answer 10

size exclusion, also known as gel filtration
ion exchange
affinity
RP-HPLC

Question 11

Based on what characteristics do the different types of chromotography seperate for proteins

Answer 11

based on teh chemical properties of teh proteins:
Size exclusion chromotography will seperate it based on sizes of the protiens
Ion exchange will use the overall charges of the proteins
Affinity will use the ligand binding of protein
RP-HPLC will use hydrophobicity

Question 12

How does the size exclusion chromotography work?

Answer 12

the proteins are seperated based on their size and the columns will contain beads which will delay smaller proteins since the proteins will get tangled with the beads and take longer to get eluted but this methods can have its downside like small proteins that are elongated being able to evade teh beads and get eluted faster

Question 13

what does void volume, eluted volume, and total volume stand for in size chromotography

Answer 13

void volume- stand for the volume which consist of mobile phase only, where there is no stationary column, beads. This volume is foudn by having very lareg molecule pass through since taht will represent teh volume available with no beads in it

eluted volume- represent teh volume of the mobile phase that must pass before a specific subtance get eluted, thsi volume is close to void voluem for large molecules but gets large for small molecules since they spend more time in due to their interaction with the beads, so more volume passes before they pass through

total volume- total volume of the column which included teh stationary and mobile phases and this can be measure by having very small molecules that can diffuse through teh pores pass through and measure teh volume required to elute it

Question 14

How does ion exchange chromotography work

Answer 14

uses charged resin as the stationary column to seperate for the protein based on its polypeptide charge
have two types to choose from: anion exchange or cation exchange
the anion exchange resin will be used to retain the - charged polypeptides meaning it is positively charged
teh cation resin exchange will be used to retain teh + charged polypeptides so it is negtaively charged

Question 15

What are the two types of resin beads column used for ion exchange chromotography

Answer 15

CM- carboxymethyl group as the negatively charged resin to attract positive polypeptides
DEAE- diethylaminoethyl which is positively charged and used to seperate the negatively charged polypeptides

Question 16

How does affinity chromotography work

Answer 16

by having the resin be covalently bound to ligand which is specific for a protein of interest which will interact with their ligand noncovalently and then the protein of interest can be released from teh resin by having another molecule passing through to compete for teh same binding so the protein of interest gets released

Question 17

What are two types of affinity chromotography

Answer 17

1- His tags and Nickel-NTA resin: where the nickel will coordinate binding of His side chains to NTA when side chain is close so His tags are added to protein to help with purification and free imidazole can be used to compete with th POI for the NTP resin and elute it
2- Immunopresipitation: use of antibodies that specifically bind to proteins where teh POI will be the antigen to bind the Fab domain on antibody found in resin and then teh POI gets percipetated out by purifying antibody on beads

Question 18

How does HPLC seperate proteins

Answer 18

using high pressure and teh resin beads are silica covered with hydrocarbons to produce high resolution of peaks

Question 19

RP-HPLC is type of HPLC, how does it work

Answer 19

this is still HPLC, but based on the hydrophobicity where hydrophobic compounds will interact stronger with column and have higher retention time

Question 20

Why is dialysis important and how is it achieved

Answer 20

dialysis is used to purify the protein from any small molecules taht were used to elute the proteins as these molecules can interfere with the experiemnt and assays
dialysis is achieved by eluted samples being placed in semipermeable dialysis bag and incubated in a buffer where diffusion will allow molecules to move from sample into buffer

Question 21

how does SDS PAGE work and what is its role

Answer 21

SDS PAGE: SDS- is sodium dodecyl sulfate which is strong detergent that denatures proetins and gives the polypeptide a uniform negative charge
PAGE- polacrylamide gel electrophoresis which creates a mesh of cross linked molecules that seperate subunits based on size
This will allow fro the protein to migrate in the gel toward anode and the size will be deduced by comparing it to MW markers
Will allow us to visualize the protein but will need further methods like mass spec or immunoblotting to figure out identity

Question 22

How is gel elctrophoresis different from chromotography

Answer 22

this will use elctric current to derive teh molecules through matrix and seperate

Question 23

What is western blotting and what is used for

Answer 23

western blotting consist of two parts, SDS PAGE and immunoblotting
SDS PAGE will help be for seperation of the polypeptide based on its MW and then immunoblotting will be used to identify the polypeptide using antibodies

Question 24

How is Western Blotting conducted

Answer 24

1- SDS PAGE to seperate the proteins based on their sizes only by providing them with unifrom - charge
2- Transfer of the proteins to polymer sheet where it is also exposed to protein solution like milk or BSA to bind random proteins to any unused regions of membrane
3- primary antibody specific for POI will be added to recognize teh linear sequence of amino cids
4- secondary antibody that is fluorescently tagged and specific for Fc domain of primary antibody is added to visualize the product

Question 25

how is mass spec conducted to identify the polypeptide

Answer 25

peptide will be hit with laser beam or high energy electron beam to create ionized fragments and then these fragements will be attracted to a charged plate deector and will be analyzed absed on tehir time of flight which depends on their mass and charge, positive and small will travel faster

Question 26

How is edman degradation conducted and what are its limitation

Answer 26

it is a chemical reaction based method that removes amino acids starting from N-terminus using proteases or chemicals and then putting these fragements together will help us identify teh polypeptide
limitations include: amino acids removed need to be purified and identified using chromotography, there is limit ot number of amino acids sequenced, and N-terminal may be modified so it cannot be removed

Question 27

what is edman degradation based on

Answer 27

it is a sequence of amino acids obtained indirecly from DNA using DNA sequencing