mcbride lecture 10: application of protein isolation techniques in diagnosis

Assay Reaction for AST

- aminotransferase reaction: formation of oxaloacetate

- AST converts a-ketoglutarate to L-glutamate in order to convert L-aspartate to oxaloacetate

indicator reaction for AST

- dehydrogenase reaction: quantitation of oxaloacetate

- Malate dehydrogenase converts oxaloacetate to L-malate at the same time it converts NADH to NADH+

analyzing purification scheme

- amount of protein m=resent is mixture being assayed must be known

- overall goal of purification is to maximize the specific activity

centrifuge step 2

- centrifuge the homogenate at low speed to yield a pellet consisting of heavy material and lighter supernatant

- separate the organelles within the cells

centrifuge step 3

- centrifuge the supernatant at a higher centrifugal force to yield another pellet and supernatant

- this process of differential centrifugation is repeated many times to yield several fractions of decreasing density

- one fraction will be enriched for desired activity

low speed centrifugation

- pellet contains whole cells, nuclei, and cytoskeletons

medium speed centrifugation

- pellet contains mitochondria, lysosomes, peroxisomes

high speed centrifugation

- pellet contains microsomes, small vesicles

very high speed centrifugation

- pellet contains ribosomes, viruses, large macromolecules

velocity sedimentation

- depends on gravity

- subcellular components sediment at different speeds according to their size and shape when layered over a solution of 5-20% sucrose

- after centrifugation, the first few fractions will have fast-sedimenting particles, whereas later fractions contain slow sedimenting particle

equilibrium sedimentation

- subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches that of their surroundings

- low-buoyant-density components are higher up in the tube

- high-buoyant-density components are found lower in the tube

salting in

- increase solubility of protein

- low level of salt will enter solution

salting out

- method of precipitating proteins for collecting

- effect by which most proteins are less soluble at high salt concentrations

salting out between different proteins

- salt concentration at which a protein precipitates differs from one protein to another

- depends on where you filter and what salt concentration the protein will precipitate out at

follow up for proteins that salted out

- centrifuge the high [salt] mixture with protein and the precipitated proteins will be in the pellet

- if your protein of interest precipitates out easily, it is in the pellet

- if protein doesn't precipitate easily, it is in supernatant

dialysis

- dialysis bag with a semipermeable membrane that separates molecules based on size, which is regulated by pore size in the membrane

- bag is placed in a saline solution(hypertonic), and small molecules that fit through pores are able to leave the bag down their concentration gradient, but the bigger ones are left in the bag

- depending on the size of your molecule, you would use what was in the bag for bigger protein or the solution for smaller protein of interest

what gel matrix usually used in column chromatography

- cellulose

gel filtration chromatography

- aka molecular exclusion chromatography

- gravity dependent

- separates proteins based on size

- column filled with porous beads which trap small molecules

beads for gel filtration chromatography

- cross-linked polysaccharide, dextran, agarose, or acrylamide

- available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various masses

basis of a blood test

- provide a snapshot of overall health and often the first step to disease diagnosis

- taken during physical or in response to some symptoms

what does blood test measure

- blood cells and platelets, electrolytes, proteins, hormones, and certain minerals

- some underlying issue can be identified based on abnormalities

examples of what blood test results could allude to

- glucose: high in diabetes

- urea: high in kidney disease

- ammonia: high when liver issue

- LDL cholesterol: high in cardiovascular disease

different proteins that blood tests quantify

- albumin

- alkaline phosphate (ALP)

- alanine aminotransferase (ALT)

- aspartate aminotransferase (AST)

- locations and functions determine their linked pathology

albumin

- marker of liver and kidney function

- also carries fatty acids

alkaline phosphatase

- high levels can indicate liver or bone disorders

alanine Aminotransferases

- high levels indicate liver damage

aspartate Aminotransferases

- high levels indicate liver damage

overall sequence of AST activity

1. assay reaction

2. indicator reaction

what can be measured to determine AST activity

- NAD+ levels

- greater means more conversion using AST, but less NAD+ means less AST activity

absorbance and NADH activity

- measuring absorbance at 340 nm indicates NADH levels

- high absorbance means more NADH, less NAD+ and overall less AST

- less absorbance means less NADH, more NAD+ and overall more AST

readouts of AST, NAD, and absorbance

- absorbance is the direct readout of NADH, so the more conversion of NADH to NAD+ correlates to lower absorbance

- conversion rate of NADH to NAD+ is an indirect readout of how much AST is present in the sample; more AST means faster rate of conversion

ALT reaction sequence

1. assay reaction

2. indicator reaction

assay reaction for ALT

- ALT converts a-ketoglutarate to L-glutamate in order to convert L-alanine to pyruvate

indicator reaction for ALT

- lactate dehydrogenase (DLDH) converts pyruvate to D-lactate at the same time it converts NADH to NADH+

absorbance of NADH at 340 nm and ALT levels

- higher absorbance means less ALT

- less absorbance means more ALT and high pyruvate and high NAD+

protein purification

- often an essential first step in their quantification and understanding their function

- performed by subjecting impure mixture of starting material to a series of separations based on physical properties like size and charge

how to verify protein purification

- requires a test/assay to determine whether the protein of interest is present

specific activity

- the ratio of enzyme activity to the amount of protein in the mixture

- high specific activity, which is the goal, is when most of the protein is the protein of interest

centrifuge step 1

- disrupt cell membranes of intact cells to form a homogenate

column chromatography

- gravity dependent and the hallmark of analytical chem

- sample of solution is placed on top of column filled with a permeable gel matrix, or any material that will interact with sample to separate it as it travels down the column

- lots of solvent passed down the column and takes the sample with it, and everything is collected in separate tubes as they elute down

interaction between matrix and elution times

- more interaction = slower elution

- less interaction = faster elution

elution times for gel filtration chromatography

- big molecules pass through faster because they don't interact with beads

- small molecules pass through slower because they get trapped in beads

graphing gel filtration chromatography

- x-axis is fraction number, where you test each fraction to see which ones have characteristics of protein

- y-axis is the relative amount

- fractions with greatest amounts of relative activity you are looking for get pooled and applied to another column

- other proteins are removed

ion-exchange chromatography

- separates protein by charge

- still based on gravity

- column filled with charged beads, and the sample is applied to the top of the column

- cation and anion exchange chromatography

cation exchange chromatography

- uses negatively-charged beads to trap positively charged ions

- ex: carboxymethyl group

- only negatively-charged proteins elute out

anion exchange chromatography

- uses positively-charged beads to trap negatively charged ions

- ex: diethylaminoethyl group

- only positively-charged proteins elute out

how are trapped proteins released in ion exchange chromatography

- ultimately released by increasing the salt concentration of the buffer that is passed through the column

- increased salt disrupts opposite interactions, which releases proteins from the grasp of bead

results of ion exchange chromatography

- protein of interest eluted in several fractions and detected by enzymatic activity

- pool fractions with most amount of activity; usually seen after the salt concentration is increased to force the elution of protein of interest

- discard remaining proteins

affinity chromatography

- takes advantage of the fact that some proteins have high affinity for specific ligands, so you must know what it is in order to utilize this method

- depends on gravity

- when protein solution is passed over the beads, proteins with affinity for the attached group are retained

- bound protein is then released by passing a solution enriched in the ligand to which the protein is bound through the column

result of affinity chromatography

- earlier fractions before an eluting solution is added are discarded because they don't have high affinity for the ligand in the column

- once eluting solution is added, all fractions collected with the protein are pooled and contain the highly purified protein

- we cannot guarantee that all the proteins are in fact the protein of interest because the same ligand can have high affinity for multiple proteins

resolving power of chromatographic technique

- related to the number of potential sites of interaction between the protein and the column beads

High-Performance Liquid Chromatography (HPLC)

- using very fine beads in columns and pressure to move the liquid through the column

- leads to sharper and more rapid separations between proteins and separations

size of beads is HPLC

- fine beads because they allow for more interaction and greater resolving power

- flow rates are slow

gel filtration by HPLC

- clearly defines the individual proteins because of greater resolving power

- absorbance X time in minutes

gel electrophoresis

- separates mixtures of molecules with a net charge by applying an electrical field

- molecules start off at the negative end and travel to the positive end

- used for separating nucleic acids and proteins

what is electrophoresis carried out in and how does that impact speed of molecules

- carried out in gels, which act as molecular sieves to enhance separation

- small molecules move quicker and farther through the gel than larger molecules

polyacrylamide gels

- highly cross-linked 3D mech

- formed by polymers of acrylamide with intermittently spaced cross-linker

- more cross links = more dense gel; varying [cross-links] can control density of gel

- density of gel allows different proteins to pass through at different rates

SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

- allows accurate determination of mass

- SDS is an anionic detergent that denatures proteins and makes it so that the 3D structure of protein has no influence on the rate that it passes through gel

- proteins have the same charge to mass ratio and migrate in the gel on the basis of mass only

SDS : AA ratio

1:2

Coomassie blue

- nonspecific staining of the protein separated by SDS-PAGE

- bonds to basic and hydrophobic amino residues

- allows visualization of number and size of protein, as well as seeing how proficiently protein purification process is going

mobility of protein in SDS-PAGE in relation to log mass

- linearly proportional

- greater mobility = decreased mass

- less mobility = increased mass

example of what sequence of proteins may be tested in SDS-PAGE

1. homogenate

2. salt fractionation

3. ion-exchange chromatography

4. gel-filtration chromatography

5. affinity chromatography

effectiveness of purification seen through SDS-PAGE

- overall gets least to most specific and effectiveness of the purification can be seen as the bad for the protein of interest becomes prominent relative to other bands

polyclonal antibodies

- heterogenous mixtures of antibodies: many antibodies for 1 antigen

- derived from multiple antibody-producing cell populations

- each antibody is specific for one of the various epitopes on an antigen

monoclonal antibodies

- homogenous antibodies produced by clones of a single antibody producing cell

- used in clinical setting

monoclonal antibody manufacturing

- can be manufactured by injecting protein into animal, isolating spleen cells, then isolate the antibodies that the cells produce and purify it

Western blotting

- proteins separated from SDS-PAGE gel, transferred to polymer, stained with a primary antibody, stained with secondary antibody and quantified through an illuminated blot

primary antibody

- antibody specific for the protein

secondary protein

- antibody specific to the primary antibody shapes

- attached to an enzyme that generates a chemiluminescent signal or fluorescent tag to enable ID and quantification

synovial sarcoma

- rare pediatric cancer driven by a chromosomal translocation that produces SS18-SSX oncogenic fusion protein

- development of antibodies targeting this fusion enables detection and quantification by western blot

almost all samples from synovial sarcoma patients have these antibodies interact with them:

- E9X9V: which binds to the fusion junction of SS18-SSX

- E5A2C: binds to C-terminus of SSX

immunohistochemistry

- accomplished with antibodies that recognize target antigen on tissue slices and are then developed to produce a dark staining where the target protein is located

Enzyme Linked Immunosorbent Assay (ELISA)

- quantifies amount of protein present in the sample that you directly add to the wells

- antibody is linked to an enzyme, such as horseradish peroxidase, that reacts with a substrate to produce colored product

- increased signal means more antibody means more antigen of interest

indirect ELISA

- antigen coated well --> add specific antibody that binds to antigen --> add enzyme linked antibody to bind to specific antibody --> substrate added to color the product

Sandwich ELISA

- type of ELISA where the antigen is pinched between 2 antibodies

- starts with the well coated with the antibody instead of the antigen

prostate specific antigen PSA

- a protein produced by prostate and plays important role in fertility

- high blood levels of PSA in prostate cancer, so this test measures PSA levels in blood by ELISA

PSA test

- is a sandwich ELISA

- each well coated with PSA-specific capture antibody

- antigen added and antibody traps it

- a biotin-conjugated detection antibody is then added, which bonds to the captured antigen

- plotted on a standard density vs concentration curve

biotin-conjugated detection antibody

- has streptavidin-HRP, which has great affinity for biotin

- increased absorbance means greater conversion of TMB by HRP = greater PSA antigen present

Co-immunoprecipitation

- enables identification of binding partners/protein complexes

Co-immunoprecipitation step 1

incubate extract with monoclonal antibody against specific protein

Co-immunoprecipitation step 2

- add agarose beads coated with an antibody-binding protein, like protein A

Co-immunoprecipitation step 3

- separate antibody bound protein cp,[;ex through centrifugation

- under optimal buffer conditions, any additional proteins bound to original protein will also precipitate

- the identity of other bound proteins can be probed by other methods like western blotting

fluorescent markers

- cells can be stained with fluorescence-labeled antibodies

- enable proteins to be visualized by fluorescence microscopy to reveal the cellular location of the protein

- provides clue to protein function

fluorescent tags on proteins

- can follow movement of proteins throughout cell

- antibodies labeled with clusters of electron dense metal can be used in immunoelectron microscopy

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