Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

molbio > Protein analysis lecture > Flashcards

Protein analysis lecture Flashcards

1
Q

a measurable substance in the blood
whose presence is indicative of diseases and environmental exposure

A

Biomarker

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Most common method for purifying proteins is

  • separation of soluble components in a solution by specific differences in physical-chemical characteristics of
    the different constituents.
  • method uses a glass or plastic tube with resin inside that
    could separate proteins
A

COLUMN CHROMATOGRAPHY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q
  • Separates molecules based on the difference in their sizes
    and shapes
  • Small molecules are more likely to go through the pores of
    the matrix and be trapped in the resin.
A

GEL FILTRATION/ SIZE
EXCLUSION

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q
  • Contains resin that is bearing either positively or negatively
    charged chemical group.
A

ION EXCHANGE CHROMATOGRAPHY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Resin that contains positively charged group

A

Anion exchange resin

Opposite attracts.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Resin that contains negatively charged groups

A

cation exchangers

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q
  • Uses the lock and key binding
  • used to separate and prepare larger quantities of proteins
    and antibodies for study.
  • It is a very efficient procedure because this method relies on
    biological specificity of the protein of interest.
A

AFFINITY CHROMATOGRAPHY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Why does protein need to bind with ligand?

A

To carry out their biological activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

o This is used for separation.
o To determine the sizes
o To determine the presence or amount of DNA.

A

ELECTROPHORESIS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q
  • Anionic detergent
  • Disrupt the structure of protein to be linear and binds most
    protein.
A

SDS-PAGE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

SDS-PAGE Denaturing agents
-Breaks the covalent bond present.

A
  • Heat
  • Dithiothreitol (DTT)
  • 2 mercapthoethanol
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

o It coats all the polypeptides with negative charges so their run in the electrophoresis will be towards
the positive charge end.

o It masks the natural charges of the sub-unit, they electrophoresis according to their molecular
masses not by their native charges.

A

2 advantages of SDS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Proteins migrate in the gel according to their molecular
masses so the electrophoretic mobility of proteins upon SDSPAGE is inversely proportional to the molecular weight.

A

PROTEIN FRACTIONATION BY SDS-PAGE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

o Coomassie Blue Staining or Coomassie
brilliant blue

o Zinc-reverse Staining

o Silver Staining

A

COLORIMETRIC

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

▪ An organic dye which is the most
frequently employed method for
protein detection in SDS-PAGE gel.

A

Coomassie Blue Staining

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

▪ Also known as negative staining

▪ It uses imidazole and zinc salts for
protein detection in electrophoresis
gels.

A

Zinc-reverse Staining

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q
  • widely used because the sensitivity
    is below one nanogram per spot and
    cost for reagents are relatively low or
    cheaper

Based on the binding of silver ions to
the proteins followed by reduction to
free silver, sensitization, and
enhancement.

A

Silver Staining

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q
  • CyeDyes
  • Nile Red
  • SyPro
  • Epicocconone
A

Fluorescence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

can even reveal as low as
1ng of protein, it is inconvenient because it has to be incorporated before gel electrophoresis

A

CyeDyes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

The sensitivities of SyPro Orange, Red, and Tangerine are similar to that of Coomassie Blue, SyPro Ruby can detect down to about 1ng of protein in a spot.

A

SyPro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Deep Purple contains the fluorophore
“epicocconone” from the fungus Epicoccum nigrum and is even more sensitive than SyPro Ruby, down to a few hundred picograms

A

Epicocconone

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

the most popular and wellestablished technique for global protein separation and quantification.

A

Gel-based proteomics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

method used to detect specific
protein molecules from among a mixture of proteins.

A

WESTERN BLOT

24
Q

Western Blot uses three steps to accomplish the goal

A
  • Seperation of Protein
  • Transferring the gel
  • Mark the target protein
25
Q

Passive transfer aided by capillary action or vacuum.

useful when the
agarose gels are cast on a plastic backing sheet.

A

Diffusion

26
Q

o Electrotransfer where the proteins are driven by an electric field.

o Electrotransfer forces the proteins out of the gel and onto the membrane by an electric field applied across the gel and the superimposed membrane.

A

Electroblotting

27
Q

essential to block the unused binding sites on the membrane.

A

blocking step

28
Q

gives optimal blocking with
lowest background and without impairing immunoreactivity of the blotted proteins.

A

Ovalbumin/gelatin

29
Q

has proved to be an economical and
effective blocker in most systems

A

fat free
milk powder

30
Q

provides a higher
sensitivity than the color production system.

A

Chemiluminescence (CL) system

31
Q
  • We can use spectrophotometers for detecting proteins
  • Amino acids with aromatic rings like tryptophan, tyrosine, and
    phenylalanine absorb UV wavelength.
A

UV ABSORPTION METHOD

32
Q
  • Buffer and reagents could interfere UV ABSORPTION METHOD with the result.
A

SDS, 2-mercaptoethanol, and TRIS Buffer

33
Q

We can modify protein samples with appropriate reagents to
produce a color reaction

A

COLORIMETRIC PROTEIN ASSAY TECHNIQUES

34
Q
  • The peptide bonds of protein react with the cupric ions
    present in biuret reagents under alkaline solution.
  • A purplish-violet color is produced, resulting from complex
    formation between the cupric ions and the peptide bond
A

BIURET METHOD

35
Q
  • This is a two-step reaction same as the biuret which serves as the first reaction.
A

LOWRY METHOD

36
Q

LOWRY METHOD Next step is the reduction of copper ions under alkaline
condition which forms a complex with peptide bond

A

cuprous complex

37
Q
  • Similar principle as Biuret except it includes BCA
  • method is based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion generated by the biuret reaction under alkaline conditions.
A

BICINCHONIC ACID METHOD

38
Q

forms a deep blue/purple color that is read at 562nm, and the detection
range is 0.2–50μg.

A

bicinchoninic acid cuprous complex

39
Q

A common laboratory technique which is used to measure
the concentration of an analyte in a solution

  • It is a quantitative immunological procedure in which antigen
    antibody reaction is monitored by enzyme measurement.
  • uses the coupling of antigens and
    antibodies and relies on the specificity and affinity of
    antibodies for antigens
A

ELISA
Enzyme Linked ImmunoSorbent
Assay

40
Q
  • It uses a primary labeled antibody that reacts directly with the
    antigen.
  • It can be performed with the antigen that is directly
    immobilized on assay plate.
  • Not widely used bit common for immune-histochemical
    staining of cells and tissues.
  • One antibody is used with enzyme links.
A

DIRECT ELISA

41
Q
  • It utilizes a primary unlabeled antibody in conjunction with a
    labeled secondary antibody
  • Secondary antibody has a specificity for primary antibody.
  • Antigen directly adsorbed onto solid phase is first incubated
    with patient serum, and then with a labeled antibody specific
    for human immunoglobulin.
A

INDIRECT ELISA

42
Q
  • Antigens like Tumor markers, hormones, serum proteins may
    be determined.
  • Antigens in the sample bind with the capture antibody and become immobilized.
  • The antibody of the enzyme conjugates binds with the
    immobilize antigen to form a sandwich of Antibody-antigenantibody enzyme bound to microwell.
A

SANDWICH ELISA

43
Q
  • Simple procedure
  • High sensitivity and specificity because of the antigen
    antibody reaction
  • High efficiency as simultaneous analysis can be
    performed without complicated sample pre-treatment.
  • Generally safe and eco-friendly because no radioactive
    substance or large number of organic solvents is
    needed that could be harmful.
A

Advantages of ELISA

44
Q
  • It is labor intensive.
  • Reagents are cheap but antibodies used is expensive.
  • You need sophisticated techniques to prepare the
    antibodies.
  • There is a high possibility of false positive or false
    negative results because insufficient blocking of the
    surface of the microtiter plate and because antibodies
    are unstable.
A

Disadvantages of ELISA

45
Q
  • used to determine the precise mass of peptides. 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. The ionized peptides in the gas are then
    accelerated in an electric field and separated.

-

A

MASS SPECTROMETRY

46
Q

MASS SPECTROMETRY BASIC OPERATIONS

A
  • Separate ions in space or time
  • Measure the quantity of the ions
47
Q

Two common method of mass spectrometry

A
  • Electrospray Ionization
  • Matrix-assisted laser desorption or ionization-time-of-flight mass spectrometry
48
Q
  • Very sensitive and accurate for determining amino acids
    sequences.
  • Peptides are first mixed with an organic acid.
  • Dry on a ceramic or metal slide or matrix
  • The sample is blasted with a laser or electron beam
    which causes the peptides to be excited or ejected from
    the slide inn a form of ionized gas.
  • The ionized peptides are then accelerated in an
    electrical field and fly towards a detector
A

MASS SPECTROMETRY

49
Q
  • Sample Volume
  • Sample recovery
  • Throughput
  • Robustness
  • Chemical modification
  • Aggregation
A

CRITERIA WHEN SELECTING AN ASSAY

49
Q
  • Sample Volume
  • Sample recovery
  • Throughput
  • Robustness
  • Chemical modification
  • Aggregation
A

CRITERIA WHEN SELECTING AN ASSAY

50
Q

What is the required measurement for each assay

A

Sample Volume

51
Q

If detection or quantification will ruin the sample

A

Sample recovery

52
Q

Amount of protein that can be produced for each sample

A

Throughput

53
Q

Repeatability

A

Robustness

54
Q

Sensitivity and specificity

A

Chemical modification

55
Q

Clamp or aggregates might block the assay and will give undersired result

A

Aggregation

molbio (17 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions