FINALS - Protein Analysis Flashcards
what is the most abundant macromolecules in the biological system
proteins
proteins are polymers comprising amino acid that are linked by ______
peptide bonds
True or False:
proteins serve as:
1. antibodies
2. enzymes
3. messengers
4. structural components
5. transport
6. transport or storage molecule
true
the proteins that can be detected in the blood of the patient are called?
biomarkers
these are measurable substance in the blood whose presence is indicative of diseases or environmental exposure
biomarkers
most common method for purifying proteins from other protein molecules with a given sample
column chromatography
this involves the separation of soluble components in a solution by specific differences in PHYSICAL-CHEMICAL CHARACTERISTICS of the different constituents
column chromatography
what does the column chromatography uses to separate proteins based on their physical properties?
glass or plastic tube with resin inside
what should be added in the column for proteins to migrate?
buffer solution
the proteins the sample of a column migrate depending on?
- nature of the matrix
- physical properties
- chemical properties
in this protein analysis technique, the resin bead has many tiny pores like a WHIFFLE BALL
gel permeation/filtration/size exclusion
in gel permeation, the molecules are separated based on their difference in?
size and shape
in gel permeation, small molecules are more likely to go through the?
pore of the matrix
true or false:
in gel filtration, large molecules move through the column more slowly since they cannot fit into the beads
false; quickly
separates proteins using resin according to their SURFACE CHARGES
ion exchange chromatography
it uses the LOCK AND KEY BINDING that is widely present in biological system
affinity chromatography
it is used to separate and prepare LARGER quantities of PROTEINS and ANTIBODIES
affinity chromatography
it uses the principle that the protein binds to molecules for which it has SPECIFIC AFFINITY
affinity chromatography
this is the specific affinity where the PROTEIN BINDS TO CARRY OUT BIOLOGICAL ACTIVITY
ligand
its purpose are for SEPARATION, to determine SIZES, PRESENCE/AMOUNT OF DNA
electrophoresis
in electrophoresis, what is used to carry out proteins with a STACKING GEL and SEPARATING GEL?
polyacrylamide gel electrophoresis (PAGE) with SDS
this technique is associated with STAINING METHOD and can DETECT BAND OF PROTEIN in a simple and relatively rapid manner
electrophoresis
this DISRUPTS the structure of protein to be LINEAR and bind most protein
sodium dodecyl sulphate (SDS)
give the agents that is used in SDS for denaturation and breaking of covalent bonds
dithiothreitol (DTT) or 2-mercaptoethanol
this is often used to determine the MOLECULAR WEIGHT of proteins
SDS
each sodium dodecyl sulfate contributes to how many negative charges?
two
can detect as low as 10 ng - 1 ng of protein bands
colorimetric
organic dye and most frequently employed method for protein detection in SDS-PAGE gel
coomassie blue staining
uses IMIDAZOLE AND ZINC SALTS for protein detection in electrophoresis gels
zinc-reverse staining/negative staining
based on the BINDING OF SILVER IONS to the proteins followed by reduction to free silver, sensitization, and enhancement
silver staining
spots in silver staining are _____ on a ____ background
dark brown to black; light beige
what is used to solve differentially sensitive proteins to silver staining?
glutaraldehyde free modified silver staining
it bands FLUORESCE
fluorescence
better resolving power as compared to the SDS-PAGE
two-dimensional gel electrophoresis
the most popular and well-established technique for GLOBAL PROTEIN SEPARATION AND QUANTIFICATION
gel-based proteomics
this is an example of a gel-based proteomics
two-dimensional gel electrophoresis
protein in 2DE is electrophoresed through a narrow tube gel containining molecules called?
ampholytes
ampholytes set up a _____
pH gradient
this is the pH at which protein has no net charge/neutral
isoelectric point
results after isoelectric focusing and SDS-PAGE are visualized and quantified through?
bioinformatic software
northern blot is for?
RNA
southern blot is for?
DNA
western blot is for?
proteins
this is a laboratory method used to detect SPECIFIC PROTEIN molecules among a MIXTURE of proteins
western blot
what solid support is used in western blotting in transferring the gel
nitrocellulose/nylonsheet/or PVDF (polyvinyl vinylidene difluoride)
this mode of transferring proteins is aided by capillary action or vacuum
diffusion/passive transfer
achieved by posing a membrane sheet on one or both side of the gel
diffusion/passive transfer
mode of transferring proteins by putting a gel membrane in a suitable holder and immersing both the gel and membrane in a tak filled with buffer fitted with TWO PLATE ELECTRODE (- and +)
electroblotting/electrotransfer
these binds to the non-specific protein binding sites to prevent the binding of the antibodies to these sites
blocking agents
these generates a colored product that could be conjugated to an enzyme
horseradish peroxidase (HRP) and alkaline phosphatase
almost same with the color formation wherein it provides HIGHER SENSITIVITY than the COLOR PRODUCTION system
chemiluminescent
in UV absorption method, what can be used in detecting proteins?
spectrophotometer
the principle of this method is that nucleic acids (DNA or RNA) contain conjugated double bonds in their purine and pyrimidine rings that have a specific ABSORPTION PEAK at 260 nm
UV absorption peak
the most commonly used reference protein in colorimetric
bovine serum albumine (BSA)
in this method the peptide bonds of protein react with CUPRIC IONS present in BIURET reagents under alkaline condition
biuret method
if cupric ions reacvct with biuret reagents it will produce a _____ color
purple
presence of protein in biuret method will produce what color
violet
this is based on the BIURET REACTION with ADDITIONAL steps and reagents to increase the sensitivity of detection
lowry protein assay
in the biuret reaction, copper interacts with four nitrogen atoms of peptides that forms ____
cuprous complex
reacts with cupric ions generated by the BIURET REACTION under ALKALINE condition forming BCA CUPROUS COMPLEX forming a purple color
bicinchonic acid method
this is a common laboratory technique which is used to MEASURE THE CONCENTRATION of an ANALYTE in a solution
ELISA
ability to DISCRIMINATE among DIVERSE protein
specificity
ability to TIGHTLY BIND to molecules
afiinity
what produces the chemical interaction between antibodies in ELISA
B cells of leukocytes and antigens
y-shaped; any foreign substances
antigen
green color attached to antigen
antibody
uses a primary labeled antibody that react DIRETCLY with the antigen
direct ELISA
it only uses an enzyme-labelled PRIMARY antibody; SECONDARY antibodies are not needed
direct ELISA
it utilizes a primary UNLABELED antibody in conjunction with a labeled SECONDARY antibody
indirect ELISA
antigens like TUMOR MARKERS, HORMONES, SERUM PROTEINS may be determined and bind with the capture antubody and become IMMOBILIZED
sandwich ELISA
uses to determine the PRECISE MASS of PEPTIDES
MALDI-TOF spectrometry
The peptides are immobilized in an organic MATRIX and then blasted with a LASER, causing them to be ejected in the form of an ionized gas.
MALDI-TOF
