TECHNIQUES IN PROTEIN ANALYSIS Flashcards
it is the most abundant macromolecules in biological system.
Proteins
They are polymers comprising amino acids that are linked by peptide bonds.
Proteins
These proteins can be detected in the blood of the patient which are considered as __________
Biomarkers
it is a measurable substance in the blood whose presence is indicative of diseases or environmental exposure
biomarkers
it is the most common method for purifying proteins from other protein molecules with a given sample
Column Chromatography
it involves the separation of soluble components in a solution by specific differences in physical-chemical characteristics of the different constituents
Column Chromatography
True or False:
Small molecules are more likely to go through the pore of the matrix. They are trapped inside the resin and will travel through the column more slowly.
True
True or False:
Large molecules move through the column more quickly
True
This type of column chromatography has many tiny pores in the resin bead.
Gel/ Gel Permeation/ Gel Filtration/ Size Exclusion
This type of column chromatography uses resin to separate proteins according to their surface charges
Ion Exchange Chromatography
It contains a resin bearing either positively or negatively charged chemical groups.
Ion Exchange Chromatography
True or False:
In Ion Exchange Chromatography, the resins with positively charged groups (anion-exchange resins) attract negatively charged solute.
True
True or False:
In Ion Exchange Chromatography, Opposite charges attract. - It traps (-) charged molecules so (+) charged
molecules are first obtained.
True
This type of chromatography uses the so called 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 for study.
Affinity Chromatography
This type of Column Chromatography It uses the principle that the protein binds to a
molecules for which it has specific affinity.
Affinity Chromatography
This technique has different purposes: : for separation, to determine the sizes, presence, or amount of DNA
Electrophoresis
We use this kind of gel electrophoresis for proteins
Polyacrylamide gel electrophoresis with SDS
It is carried out with a stacking gel and separating gel
Polyacrylamide gel electrophoresis with SDS
This technique associated with staining method can detect bands of protein in a simple and relatively rapid manner.
Electrophoresis
SDS-PAGE meaning
Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis
True or False:
Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis is an anionic detergent (-)
True
It disrupts the structure of protein to be linear and binds most protein
Sodium Dodecyl Sulphate
These agents break the covalent bonds that are present
dithiothreitol (DTT) or 2-
mercaptoethanol
Advantages of SDS
it coats all the polypeptides with negative charges
It masks the natural charges of the subunit
What are the two types of staining methods
Colorimetric and Fluorescence
Give the 3 high sensitivity colorimetric staining methods that can be used either directly after electrophoresis
Coomassie blue staining, zinc-reverse staining, and silver staining
It is one of the 3 high sensitivity colorimetric that is most frequently employed method for protein detection in SDS-PAGE gel
Coomassie blue staining
It is one of the 3 high sensitivity colorimetric that Uses imidazole and zinc salts for protein
detection in electrophoresis gels
Zinc-reverse staining/ Negative Staining
It is one of the 3 high sensitivity colorimetric that is based on the binding of silver ions to the
proteins followed by reduction to free silver, sensitization, and enhancement
Silver staining
This dye give very wide linear dynamic ranges, over four orders of magnitude.
Fluorescence dye
Although this type of dye can even reveal as low as 1 ng of protein, it is inconvenience because it has
to be incorporated before gel electrophoresis.
CyeDyes
It is a better resolving power as compared to the SDSPAGE, especially if there is a mixture of polypeptides or when it’s too complex
Two-dimensional Gel Electrophoresis
the complex protein samples are
separated in two dimensions according to their net charge at different pH and their molecular
weights determined by SDS-PAGE.
Two-dimensional Gel electrophoresis
In this dimension, the . Sample is placed in a narrow tube gel or subjected to isoelectric focusing
based on pH.
First Dimension
In this dimension, the strip is placed at the top of the SDS-PAGE.
Second dimension
It is another method related to gel electrophoresis that is used to detect specific protein molecules from among a mixture of proteins
Western Blot
This blot is used to detects the RNA
Northern blot
This blot is used to detects the DNA
Southern blot
This blot is used to detects the Proteins
Western blot
3 elements used in western blot
- Separation by size.
- Transfer to a solid support.
- Use a specific or proper primary and secondary antibody for visualization.
It is a method of transferring protein onto membranes that is aided by capillary action (by placing a weight on the gel and membrane) or vacuum (for faster and more uniform transfer)
diffusion/ passive transfer
It is achieved by posing a membrane sheet on one or both side of the gel
diffusion/ passive transfer
This method of transferring protein onto membranes are driven by an electrical field and is achieved by putting a gel membrane in a
suitable holder and immersing both the gel and membrane in a tank filled with buffer fitted
with two plate electrode (+ and -)
electroblotting/ electrotransfer
This method of transferring protein can use spectrophotometer in detecting proteins
UV Absorption Method
A common laboratory technique which is used to measure the concentration of an analyte in a solution
ELISA
It is a quantitative immunological procedure in which antigen-antibody reaction is monitored by
enzyme measurement.
ELISA
This assay uses the coupling of antigens and antibodies and relies on the specificity and affinity of antibodies for antigens.
ELISA
It uses a primary labeled antibody that react directly with the antigen
DIRECT ELISA
It can be performed with the antigen that is directly immobilized on assay plate.
DIRECT ELISA
it uses only an enzyme-labelled
primary antibody. The secondary antibodies are not needed.
DIRECT ELISA
It utilizes a primary unlabeled antibody in conjunction with a labeled secondary antibody
INDIRECT ELISA
both primary and secondary
antibody are used, but in this case, the primary antibody is not labelled (no enzyme-linked). The enzyme is labelled with the secondary
antibody.
INDIRECT ELISA
Antigens like Tumor markers, hormones, serum proteins may be determined.
SANDWICH ELISA
In this type of ELISA, you need to prepare a surface to which a known quantity of antibody is bound.
SANDWICH ELISA
it is Used to determine the precise mass of peptides.
MALDI-TOF
IT is a very sensitive and accurate for determining amino acid sequences.
MALDI-TOF
