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Molecular Biology Lecture > 1: Techniques in Protein Analysis (FINALS) > Flashcards

1: Techniques in Protein Analysis (FINALS) Flashcards

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1
Q

→ The most abundant macromolecules in biological system

→ polymers comprising amino acids that are linked by peptide bonds

→ are diverse in their chemical and physical properties

→ serve as antibodies, enzymes, messengers, structural components, transport, transport or storage molecule

A

Proteins

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2
Q

Proteins are polymers comprise of what?

A

Amino acids

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3
Q

Proteins are amino acids linked by what?

A

Peptide bonds

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4
Q

T or F

many diseases can be distinguished from others based on the type of proteins

A

T

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5
Q

These proteins can be detected in the blood of the patient

A

Biomarkers

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6
Q

A measurable substance in the blood whose presence is indicative of
diseases or environmental exposure

A

Biomarkers

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7
Q

When can proteins be further purified? (2 conditions needed)

A
  1. After cells are broken
  2. Cell extracts are released
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8
Q

The most common method for purifying proteins from other protein molecules with a given sample

A

Column Chromatography

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9
Q

Involves the separation of soluble components in a solution by specific differences in physical- chemical characteristics of the different constituents

A

Column Chromatography

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10
Q

T or F

Separation of soluble components in solution through column chromatography happens due to the similarities of the physical-chemical characteristics

A

F (DIFFERENCES of the physical-chemical characteristics)

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11
Q

Column Chromatography uses what equipment?

A

glass or plastic tube with resin

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12
Q

Familiarize the method of column chromatography

A
  1. Protein sample is applied to the top of the column
  2. Buffer solution is flowed continuously through the column
  3. Proteins in sample migrate through column
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13
Q

In the Column Chromatography method, proteins in sample migrate at different rate due to what factors? (clue: tatlo ‘to)

A
  1. Nature
  2. Physical properties
  3. Chemical properties
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14
Q

T or F

Proteins in the sample migrate through the column at different rate depending on the nature of the matrix and the physical and chemical properties of the protein.

A

T

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15
Q

What are the 3 types of Column Chromatography method?

A
  1. Gel/Gel Permeation/
    Gel Filtration/Size Exclusion
  2. Ion Exchange Chromatography
  3. Affinity Chromatography
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16
Q

3 Types of Column Chromatography:

In this type of column chromatography, the resin bead has many tiny pores like a whiffle ball

A

Gel/Gel Permeation/
Gel Filtration/Size Exclusion

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17
Q

3 Types of Column Chromatography:

In this type of column chromatography, it separates molecules based on difference in their size and shape.

A

Gel/Gel Permeation/
Gel Filtration/Size Exclusion

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18
Q

Gel/Gel Permeation/
Gel Filtration/Size Exclusion Type of Column Chromatography:

Which are more likely to go through the pore of the matrix, the SMALL or LARGE molecules?

A

Small molecules

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19
Q

Gel/Gel Permeation/
Gel Filtration/Size Exclusion Type of Column Chromatography:

T or F

Small molecules move through the column more quickly since they cannot fit into the beads. - They are excluded from entering the pores of the beads

A

F (LARGE MOLECULES move through the column more quickly since they cannot fit into the beads. - They are excluded from entering the pores of the beads)

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20
Q

T or F

In Gel/Gel Permeation/
Gel Filtration/Size Exclusion, the first size of molecules to be eluted/separated are the large molecules

A

T

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21
Q

3 Types of Column Chromatography:

In this type of column chromatography, it uses resin to separate proteins according to their surface charges

A

Ion Exchange Chromatography

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22
Q

T or F

The resin in Ion Exchange Chromatography contains only positively charged chemical groups

A

F (contains EITHER POSITIVE OR NEGATIVELY CHARGED CHEMICAL GROUPS)

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23
Q

3 Types of Column Chromatography: Ion Exchange Chromatography

The concept to remember in Ion Exchange Chromatography?

A

Opposite charges attract

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24
Q

3 Types of Column Chromatography: Ion Exchange Chromatography

Anion-exchange/Resins with positively charged groups resins attract what?

A

negatively charged solute

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25
Q

Column chromatography: Ion exhange

Cation-exchange/Resins with negatively charged groups resins attract what?

A

positively charged solute

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26
Q

T or F

Anion-exchange resins

In low-salt solutions, it will compete or bind with negatively charged resin

A

T

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27
Q

T or F

Anion-exchange resins

In high-salt solutions, it will compete or bind with positively charged resin so negatively charged resin can be eluted

A

T

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28
Q

Ion Exchange Chromatography: Anion-exchange resins

Low salt = bind with __________

High salt = bind with ___________ and elute __________

A

Low salt = bind with (-) charged resin

High salt = bind with (+) charged resin and elute (-) charged proteins

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29
Q
A
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30
Q

3 Types of Column Chromatography:

→ uses the so called lock and key binding

→ separate and prepare larger quantities of proteins and antibodies for study

→ uses the principle that the protein binds to a molecules for which it has specific affinity

A

Affinity Chromatography

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31
Q

3 Types of Column Chromatography: Affinity Chromatography

The specific affinity where the protein
binds to carry out their biological activity

A

Ligand

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32
Q

What happens to the sample once the ligand is bound to the protein?

A

protein of interest is removed from the mixture and eluted from the resin

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33
Q

TOF. The Affinity Chromatography method relies on artificial specificity of the protein of interest, it is a very
efficient procedure.

A

F (biological specificity)

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34
Q

Electrophoresis

What gel do we use for proteins?

A

polyacrylamide gel
electrophoresis (PAGE) with SDS

stacking gel and separating gel

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35
Q

this technique associated with staining method
can detect bands of protein in a simple and
relatively rapid manner

A

polyacrylamide gel electro (PAGE) with SDS

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36
Q

the very first qualities (2) that need to be assessed for any protein sample that is routinely achieved by SDS-PAGE.

A

integrity and purity

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37
Q

Sodium Dodecyl Sulphate (SDS)

TOF. Disrupts the structure of protein to be linear and binds most DNA.

A

F (most protein)

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38
Q

other than denaturation through SDS, this other reducing agent utilized to make proteins linear.

A

Dithiothreitol (DTT) or mercaptoethanol

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39
Q

TOF. The number of SDS molecules bound by a polypeptide is proportional to the length (the number of amino acid residues) of the polypeptide.

A

T

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40
Q

What part of the SDS interacts strongly with polypeptide chains

A

hydrophobic tail

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41
Q

TOF. Each sodium dodecyl sulfate contributes one negative charges

A

F (two)

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42
Q

SDS

TOF. One of the disadvantage of SDS is that it masks the natural charges of the subunit.

A

F (advantage)

it allows them to electrophorese according to their molecular masses and not by their native charges

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43
Q

Protein Fractionation by SDS-PAGE

TOF. After the reduction and denaturation by SDS, proteins migrate in the gel according to their molecular mass.

A

T

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44
Q

TOF. The electrophoretic mobility of proteins upon
SDS-PAGE is directly proportional to the molecular weight.

A

F (inversely)

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45
Q

TOF. number of SDS molecules bound by a
polypeptide is proportional to the length of the
polypeptide.

A

T

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46
Q

What is added to make the surroundings become negatively charged and covalent bond or disulfide bond is broken between the two subunits for them to become linear?

GEL ELECTROPHORESIS

A

SDS and mercaptoethanol,

they migrate based on molecular mass, not charges (for protein fractionation)

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47
Q

Staining Methods

for Electrophoresis (2)

A

Fluorescence and colorimetric

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48
Q

Staining method

They can detect as low as 10 ng – 1 ng of protein bands.

A

colorimetric

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49
Q

Staining Methods

3 high sensitivity colorimetric staining methods

A
  1. Coomassie blue staining
  2. Zinc-reverse staining/Negative Staining
  3. Silver staining
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50
Q

Staining Methods

  • organic dye
  • most frequently employed method for protein detection in SDS-PAGE gel
A

Coomassie blue staining

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51
Q

Staining Methods

  • quantitative binding of the dye to proteins
  • long staining time
  • low sensitivity
  • narrow dynamic range
  • good reproducibility (easier to repeat)
A

Coomasie blue staining

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52
Q

Zinc-reverse staining/Negative Staining

uses what for protein
detection in electrophoresis gels?

A

imidazole and zinc salts

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53
Q

Visualization of GE: Colorimetric

rapid, simple, cheap, reproducible, compatible with mass spectrophotometer (MS) analysis

A

Zinc-reverse staining

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54
Q

Silver staining

This is based on the binding of silver ions to the proteins followed by?

A

reduction to free
silver, sensitization, and enhancement

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55
Q

Staining Method

  • widely used because the sensitivity is below 1 ng per spot
  • cheaper
  • more rapid turnaround time
A

Silver staining

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56
Q

Silver staining

spots are color?

A

dark brown to black on a
light beige background

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57
Q

Staining method

proteins are differentially
sensitive is a disadvantage that belongs to?

A

silver staining

cannot be further used for MS analysis

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58
Q

Silver staining

When proteins are differentially sensitive, we may use?

A

glutaraldehyde free modified silver staining

compatible in MS analysis

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59
Q

Fluorescence

Which does not belong:
A. CyeDyes
B. GreenBYR
C. Nile Red
D. SyPro
E. NOTA

A

B

+ Epicocconone

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60
Q

Better resolving power as compared to the SDS-PAGE, especially if there is a mixture of polypeptides or when it’s too complex (proteomics)

A

Two-Dimensional Gel Electrophoresis

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61
Q

Two-Dimensional Gel Electrophoresis

This gel is the most popular and well-established technique for global protein separation and quantification

A

Gel-based proteomics

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62
Q

gel-based proteomics

TOF. overall protein expression of tissues can be analyzed on a large
scale, and it is a cheaper approach than gel-free proteomics.

A

T

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63
Q

2 dimensional gel electrophoresis

complex protein samples are separated in two
dimension, those are?

A
  1. subjected to isoelectric
    focusing
  2. place the narrow tube gel on the top of the SDS-PAGE
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64
Q

Two-Dimensional Gel Electrophoresis

The protein is electrophoresed through a narrow tube gel containing molecules called?

A

Ampholytes

crucial to set up a pH gradient

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65
Q

Two-Dimensional Gel Electrophoresis

The migrating protein towards the increasing pH will only stop when?

A

reaches the isoelectric point

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66
Q

Two-Dimensional Gel Electrophoresis

The pH at which protein has no net charge/neutral

A

isoelectric point

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67
Q

Two-Dimensional Gel Electrophoresis

After the first dimension, what happens to the protein sample on the narrow tube gel?

A

It will be placed on the top of the SDS-PAGE

Then, SDS page will separate the proteins based on their sizes.

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68
Q

Two-Dimensional Gel Electrophoresis

They are visualized and quantified through?

A

bioinformatic software

spot visualization, spot evaluation and expression analysis

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69
Q

Two-Dimensional Gel Electrophoresis

After protein has been detected, separated, and quantified, it will be followed by the identification of protein via?

A

MS analysis

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70
Q

Western Blot

Methods of Transferring Protein into membrane?

A
  1. Diffusion/Passive Transfer
  2. Electroblotting
  3. UV absorption method
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71
Q

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

A

Western Blot

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72
Q

Western Blot

The separated proteins are then transferred
(blotted) to?

A

nitrocellulose or nylon sheet

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73
Q

Western blot

Elements, except:
A. Electrophoresed using ampholytes
B. Transfer the gel to a solid support
SDS-PAGE
C. Primary and secondary antibody
D. Separation by sizes/molecular mass using

A

A

AGAIN:
1. Separation by size.
2. Transfer to a solid support.
3. Use a specific or proper primary and secondary antibody for visualization.

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74
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Diffusion/Passive Transfer is aided by?

A

capillary action or vacuum

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75
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Achieved by posing a membrane sheet on one
or both side of the gel.

A

Diffusion/Passive Transfer

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76
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

the transfer is not efficient with
polyacrylamide gel, but it works well with agarose gel because of larger force

A

diffusion

77
Q

Western blot: Diffusion/Passive Transfer

TOF. To increase the speed of transfer, one can create a steady flow of the buffer through the gel and membrane.

A

T

78
Q

Western blot: Diffusion/Passive Transfer

TOF. Diffusion emthod advantage is it is efficient with polyacrylamide gel

A

F

transfer in diffusion method IS NOT EFFICIENT WITH POLYACRYLAMIDE GEL

works well only on agarose (large molecules)

79
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

→Proteins are driven by an electrical field

→ 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 -)

A

Electroblotting

80
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

To increase trasnger efficiency, place a stack of wet filter paper with transfer buffers on one side of the gel membrane sandwich.

Electroblotting

A

F (place a stack of wet filter paper with transfer buffers on BOTH SIDES of the gel membrane sandwich.

81
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Advantage of elctroblotting?

A

fast and gives sharp replica of the gel

82
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

disadvntages of electroblotting?

A

since electrical force is used, some proteins may not be retained by the membrane

83
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

How to fix disadvantage of electroblotting>

some proteins may not be retained by the membrane

2 ‘to

A
  1. know the right adjustment for the protein to
    bind to the membrane
  2. Optimize the electrical voltage/force
84
Q

Familiarize the steps of Electroblotting

Methods of Transferring Protein onto Mebranes (Western Blot)

A
  1. Load protein sample and apply current
  2. Transfer to solid support/gel membrane
  3. Block unused binding sites on the membrane
  4. Add specific antibodies for visualization (through incubating)
  5. Washing
  6. Visualization through autoradiography/chemiluminescent (depends on how antibody was labelled)
85
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

After adding specific antibodies, it is essential to block unused binding sites on membrane

Electroblotting

A

F (BLOCK the unused binding sites on the membrane ** BEFORE** adding specific antibodies)

86
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

What do you use to block membrane?

Electroblotting

A

Incubate with Neutral protein (BSA, milk, ovalbumin)

87
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

Purpose of blocking agents is to bind the non- specific protein binding sites, to prevent the binding of the antibodies to these sites.

Electroblotting

A

T

88
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

Blocking of antibodies improves specificity of the assay

Electroblotting

A

F (improves SENSITIVITY of the assay)

89
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Step in electroblotting done after blockig wherein protein is reacted with the labeled antibody

A

Incubation with labeled antibody

90
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

Unreacted antibody is washed away

Electroblotting

A

T

91
Q

Electroblotting: Detection

T or F

method of visualization depends on how the antibody was labelled

A

T

92
Q

Electroblotting: Detection

2 of Methods of visualization for electroblotting?

A
  1. Radiolabelling (Autoradiography)
  2. Chemiluminescent (Photographic film)
93
Q

Electroblotting: method of Detection

Almost same with the color formation wherein it provides higher sensitivity than the color production system.

A

Chemiluminescent

94
Q

Electroblotting: method of Detection

Almost same with the color formation wherein it provides higher sensitivity than the color production system.

A

Chemiluminescent

95
Q

Electroblotting: method of Detection (chemiluminescent)

The enzyme activates a substrates for what substances?

di ko gets to

A

alkaline phosphatase and cyclic diacyl hydracide

96
Q

Electroblotting: method of Detection (chemiluminescent)

alkaline phosphatase and cyclic diacyl hydracide is used for what?

di ko gets to

A

HRP (horseradish peroxidase).

97
Q

Electroblotting: method of Detection (chemiluminescent)

How is signal recorded in this visualitzation method?

A

THROUGH photographic film

98
Q

Electroblotting: method of Detection (chemiluminescent)

Advantage of chemiluminescent as a method of detection?

A

Quantitative determination of the protein by densitometry to detect and quantify the protein.

99
Q

Electroblotting: method of Detection (chemiluminescent)

T or F
In using densitometry, manual detection is needed for encoding data

Advantages of using cheiluminescent

A

F (In using densitometry, SOFTWARE is needed for encoding the data.

100
Q

Electroblotting: method of Detection (chemiluminescent)

Disadvantage of chemiluminescent as a method of detection ?

A

Knowing right exposure time for film to clearly visualize the protein transferred or detected on photographic film.

101
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

→ The amino acids with aromatic rings like tryptophan, tyrosine, phenylalanine absorb UV wavelength.

→ quantification of proteins

A

UV absorption/Spectrophotometer

102
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

What 3 amino acids with aromatic rings absorb UV length AND and how strong absorption at UV 280 nm

UV absorption/Spectrophotometer

A

tryptophan, tyrosine, phenylalanine (TTP)

103
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

Amino acids with aromatic rings show strong absorption at UV 290 nm especially tryptophan and tyrosine

UV absorption/Spectrophotometer

A

F (strong absorption at UV 280 nm especially tryptophan and tyrosine.

104
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

since absorption is directly proportional to the concentration, therefore spectrophotometer is useful to qualitate the protein concentration.

UV Absorption Method

A

F (since absorption is directly proportional to the concentration, therefore spectrophotometer is useful to quantitate the protein concentration)

105
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Advantages of uv absorption?

UV absorption

A

fast, sensitive, and easily automated.

106
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Disdvantages of uv absorption?

UV absorption

A

→ If the protein does not contain amino acid, it will not absorb UV light

→ Buffer, reagents, and nucleic acids (DNA/RNA) may interfere with results.

107
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Preferred absorbance if protein concentration is lower (around 1-100
microgram per ml).

UV absorption

A

205nm absorbance

108
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

In every 280nm, you need 20-3,000 of micrograms of protein

UV absorption

A

T

109
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

UV absorption peak of purine and pyrimidine rings

UV absorption

A

260 nm

pero sabi kanina 280 nm i acyually dk

110
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

Determination of protein concentration by measurement of absorbance at how much nm?

UV absorption

A

205 nm (A205)

111
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

Determination of protein concentration by measurement of absorbance at 205 nm (A205) is based on absorbance by the H bond.

UV absorption

A

F (Determination of protein concentration by measurement of absorbance at 205 nm (A205) is based on absorbance by the PEPTIDE BOND)

112
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

UV absorption assay can be used to quantitate protein solutions with concentrations of 2–200μg/mL protein.

UV absorption

A

F (1–100μg/mL protein)

UV absorption

113
Q

Methods of Transferring Protein onto Mebranes (Western Blot)

T or F

The quantitation of proteins by peptide bond absorption at 205 nm is more universally applicable than A280.

UV absorption

A

T

114
Q

Technique in Protein Analysis

→ This technique enable us to modify protein sample with appropriate reagents to produce a color reaction

→ After the color reaction, you may measure the
protein concentration using a spectrophotomete

→ Can either be qualitative or quantitative

A

Colorimetric Protein Assay Techniques

115
Q

Colorimetric Protein Assay Techniques

T or F

Colorimetric Protein Assay Techniques can be:

qualitative after using spectrophotometer

and

quantitative is there is color formation

A

F (baliktad my love; quali if color formation, quanti after spectrophotometer)

116
Q

Colorimetric Protein Assay Techniques

Colorimetric requires this to estimate the concentration of a sample.

A

protein standard

117
Q

Colorimetric Protein Assay Techniques

What protein standard or reference protein is commonly used?

A

Bovine serum albumin (BSA)

118
Q

Colorimetric Protein Assay Techniques

Before using colorimetric what following things should be prepared first?

A
  1. Stock solution for reference protein
  2. Construct calibration curve or reference protein
  3. Determine unknown protein using constructured calibration curve (compare unknown to reference)
119
Q

Colorimetric Protein Assay Techniques

T or F

The ideal protein standard to use in a quantitative assay is different from a matched matrix/solution that has been assigned using a higher order method (for example AAA or gravimetric analysis)

A

F (The ideal protein standard to use in a quantitative assay is the EXACT SAME PROTEIN in a matched matrix/solution that has been assigned using a higher order method)

120
Q

UV Absorption Method

Three methods for Colorimetric Technique?

A
  1. Biuret method
  2. Lowry method
  3. Bicinchonic Acid Method (BCA Method)
121
Q

Three methods for Colorimetric Technique

→ In here, the peptide bonds of protein react with cupric ions present in reagents under alkaline condition.

→ produce purple result

A

Biuret method

122
Q

Three methods for Colorimetric Technique: Biuret method

If cupric ions react with biuret reagents, it will produce what color?

A

Purple

123
Q

Three methods for Colorimetric Technique: Biuret method

You can quantify/detect it under the absorbance?

nm

A

540nm.

124
Q

Three methods for Colorimetric Technique:

somewhat insensitive compared with the other methods of colorimetric protein determination.

disadvantage

A

Biurret method

125
Q

Three methods for Colorimetric Technique:

→ two-step reaction
→ Folin-Ciocalteu reagents is added to increase the sensitivity
→ based on the biuret reaction with additional steps and reagents to increase the sensitivity of detection
→ produce blue-green results

A

Lowry method

126
Q

Three methods for Colorimetric Technique: lowry method

2 steps reaction of Lowry method

A
  1. Bind with cupric ions
  2. Reduction of copper ions under alkaline condition which form a complex with peptide bond (cause color change)
127
Q

Three methods for Colorimetric Technique: lowry method

T or F

Lowry Method’s difference from Biuret Method is that there is a second reaction which is the reduction of copper ion from Folin-Ciocalteu reagent by the copper peptide bond complex.

A

T

128
Q

Three methods for Colorimetric Technique: lowry method

What reagent is used added to increase the sensitivity

A

Folin-Ciocalteu

129
Q

Three methods for Colorimetric Technique: lowry method

Folin-Ciocalteu reagents is added to increase the sensitivity. Commonly, this method is called as the?

A

“Phosphomolybdic/Phosphotungstic Method”.

130
Q

Three methods for Colorimetric Technique: biuret

T or F

In the biuret reaction, copper interacts with five nitrogen atoms of peptides to form a cuprous complex.

A

F (FOUR NITROGEN atoms)

131
Q

Three methods for Colorimetric Technique:

→ two-step reaction
→ React with cupric ions generated by the biuret reaction under alkaline condition forming a BCA cuprous complex forming a purple color
→ color changes overtime

A

Bicinchonic Acid Method (BCA Method)

132
Q

Three methods for Colorimetric Technique:

BCA is based on the fact that the ________ reacts with the _______

A

based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion

133
Q

Three methods for Colorimetric Technique:

T or F

The bicinchoninic acid cuprous complex forms a deep blue color that is read at 562 nm, and the detection range is 0.2–50μg.

A

T

134
Q

Protein Technique Analysis

Known as ELISA

A

Enzyme-Linked Immuno Sorbent Assay

135
Q

Protein Technique Analysis

→was introduced by Peter Perlmann and Eva Engvall at Stockholm University in Sweden

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

→ quantitative immunological procedure in which antigen-antibody reaction is monitored by enzyme measurement.

A

Enzyme-Linked Immuno Sorbent Assay (ELISA)

136
Q

Protein Technique Analysis: ELISA

T or F

ELISA is a quantitative immunological procedure

A

T

137
Q

Protein Technique Analysis: ELISA

ELISA relies on what concept?

A

specificity and affinity of antibodies for antigens

138
Q

Protein Technique Analysis: ELISA

ability to discriminate among diverse protein

A

Specificity

139
Q

Protein Technique Analysis: ELISA

ability to tightly bind to molecules

A

Affinity

140
Q

ELISA

is based on the concept of antigen-antibody reactions, representing the chemical interaction between antibodies produced by the _________________________ of _______________________ and ______________________

A

B cells of leukocutes and antigens

141
Q

Protein Technique Analysis: ELISA

This specific immune response plays an important role in protecting the body from invaders such as pathogens and toxins.

A

antigen-antibody reaction

142
Q

Protein Technique Analysis: ELISA

Familiarize the general principle of ELISA

A
  1. In ELISA, a specific antibody is coupled to a solid support.
  2. Samples to be assayed are added to the wells (antibody added)
  3. Wells are washed to remove the unbound antigens or unbound proteins.
  4. Second antibody is added. This antibody recognizes the same protein or the same antigen.
  5. The secondary antibody is attached to an enzyme (catalyze the conversion from colorless to colored product)
143
Q

Protein Technique Analysis: ELISA

Convenient format for ELISA?

A

use plastic tray that has molded wells or microtiter tray (preferred solid support).

144
Q

Protein Technique Analysis: ELISA

T or F

Samples may contain antibody like proteins which is recognized by antigen

A

F (reverse; Samples may contain ANTIGEN like proteins which is then recognized by ANTIBODIES)

145
Q

Protein Technique Analysis: ELISA

If there’s antigen in the sample = ??

A

If there’s antigen in the sample = attach to antibody

146
Q

Protein Technique Analysis: ELISA

After binding of antigen with antibody, the wells are what?

A

Washed to remove the unbound antigens or unbound proteins.

147
Q

Protein Technique Analysis: ELISA

After binding, washing, what is done next

4th step

A

Second antibody is added

148
Q

Protein Technique Analysis: ELISA

T or F

Second antibody happens on same binding site than the first antibody.

A

F (happens on DIFFERENT binding site than the first antibody.

149
Q

Protein Technique Analysis: ELISA

To what does the secondary antibody attaches to that catalyzes the conversion of a colorless or non-fluorescence substrate into a colored or fluorescent product.

A

Enzyme

150
Q

Protein Technique Analysis: ELISA

is then measured to determine the amount of antigen present in each sample.

A

intensity of the color or the fluorescence produced

151
Q

Types of ELISA ?

A
  1. Direct ELISA
  2. Indirect ELISA
  3. Sandwich ELISA
152
Q

Types of ELISA

→ uses a primary labeled antibody that react directly with the antigen

→ It can be performed with the antigen that is directly immobilized on assay plate.

→ Not widely used but common for immuno- histochemical staining of cells and tissues.

A

DIRECT ELISA

153
Q

Direct, Indirect, Sandwich ELISA

→ only uses an enzyme-labelled primary antibody

A

DIRECT ELISA

154
Q

Direct, Indirect, Sandwich ELISA

→ common for immuno- histochemical staining of cells and tissues.

A

DIRECT ELISA

155
Q

Types of ELISA: DIRECT ELISA

Familiarize steps of ELISA

A
  1. The enzyme-labelled (antigen) is immobilized to the plate or solid surface.
  2. The enzyme-linked primary antibody attaches to the antigen.
  3. It will then react with the substrate to produce a visible signal that can be measured.
156
Q

Direct, Indirect, Sandwich ELISA

the antigen of interest is detected.

A

DIRECT ELISA

157
Q

Types of ELISA

→ 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

158
Q

Direct, Indirect, Sandwich ELISA

Unlabelled primary and labelled secondary antibody is used

A

Indirect ELISA

159
Q

Types of ELISA: INDIRECT ELISA

is directly adsorbed onto solid phase is first incubated with patient serum and then with a labeled antibody specific for human immunoglobulin.

A

Antigen

160
Q

Types of ELISA

→ 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 conjugate bind with the immobilized antigen to form a sandwich of Ab-Ag-Ab/ enzyme bound to microwell.

A

SANDWICH ELISA

161
Q

Types of ELISA: SANDWICH ELISA

(1) This bind with the capture antibody and become immobilized.

A

Antigens in the sample

162
Q

Types of ELISA: SANDWICH ELISA

(2) This bind with the immobilized antigen to form a sandwich of Ab-Ag-Ab/ enzyme bound to microwell

A

Antibody of the enzyme conjugate

163
Q

Types of ELISA: SANDWICH ELISA

Familiarize steps of SANDWICH ELISA

A
  1. Prepare a surface to which a known quantity of antibody is bound.
  2. Add the antigen-containing sample to the plate (will bind to the antibody is specific–forming antigen-antibody complex)
  3. Incubate at 37°C and wash the plate to remove the unbound antigens.
  4. Add the enzyme-linked antibody (specific to the antigen)
  5. Incubate at 37°C and wash the plate to remove the unbound antigens.
164
Q

Direct, Indirect, Sandwich ELISA

Enzyme reacts with the substrate and it will then be converted into a colored product or fluorescence.

A

Sandwich ELISA

lahat naman ata nagcocolor change

165
Q

Types of ELISA: INDIRECT ELISA

T or F

Color change is inversely proportional to the concentration of specific antibodies in the specimen

A

F (Color change is DIRECTLY proportional to the concentration of specific antibodies in the specimen)

166
Q

Direct, Indirect, Sandwich ELISA

The enzyme links to the secondary antibody reacts to the substrate to produce a visible signal that can be measured.

A

Indirect ELISA

SANDWICH is also has an enzyme-linked antibody (secondary)

167
Q

ELISA

Familiarize advantages of ELISA

A

→ Simple procedure

→High specificity and high sensitivity because of
the antigen-antibody reaction.

→ Has high efficiency as simultaneous analysis can be performed since it has many plates without complicated sample pre-treatment.

→Generally safe and eco-friendly because radioactive substance or large amount of organic solvents are not needed.

168
Q

ELISA

Familiarize disadvantages of ELISA

A

→ Labor intensive many steps to be followed. You will just follow the protocols/steps indicated in the leaflets attached to the kits.

→ Antibodies to be used are pricey and it needs sophisticated techniques to prepare it.

→ High possibility of false positive or false negative results because of insufficient blocking of the surface of the microtiter plate and because antibodies are unstable.

169
Q

Identify corresponding processes:

Separation of proteins: (2)
Quantification of proteins: (2)
Identifying: (1)

A

Separation of proteins: Column Chromatography, Gel Electrophoresis

Quantification of proteins: Western BLOT, ELISA

Identifying: Mass Spectrometry

170
Q

Mass Spectrometry

Basic steps in Mass Spectrometry

A
  1. Separate the ions and space or time
  2. Measure the quantity of the ions.
171
Q

Mass Spectrometry

Two most common methods in using Mass Spectrometry

A
  1. Electrospray ionization
  2. MALDI-TOF
172
Q

Methods in using Mass Spectrometry:

→ Used to determine the precise mass of peptides

→ 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.

→ ionized peptides in the gas are then accelerated in an electric field and separated.

A

Matrix-assisted laser desorption ionization- time-of-flight (MALDI-TOF) spectrometry

173
Q

Identify if the process for MALDI-TOF is true or false

T or F

  1. Ionized peptides in the gas are then accelerated in an electric field and separated.
  2. 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.
A

F (baliktad)

174
Q

Methods in using Mass Spectrometry: MALDI-TOF

are immobilized in an organic matrix and then blasted with a laser, causing them to be ejected in the form of an ionized gas.

A

Peptides

175
Q

Methods in using Mass Spectrometry: MALDI-TOF

Peptides are immobilized in an organic matrix and then blasted with a laser, causing them to be ejected in the form of an ?

A

Ionized gas

176
Q

very sensitive and accurate for determining amino acid sequences

A

MALDI-TOF

177
Q

MALDI-TOF

Familiarize steps in MALDI-TOF

A
  1. Peptides are mixed with an organic acids and then dried onto a ceramic/metal slide
  2. When the sample (analyte) is stabilized in the slide, it will then be blasted with a laser or electron beam which causes the peptides to be excited or ejected from the slide in the form of an ionized gas.
  3. These ionized peptides are then accelerated in an electrical field and fly towards a detector for the proteins to be identified.
178
Q

MALDI-TOF detection process

T or F

The time it takes for the ions to reach the detector is determined by their mass

A

F (MASS AND CHARGE)

179
Q

MALDI-TOF detection process

The velocity of the attracted ions is determined by what law?

A

Law of conservation of energy

180
Q

MALDI-TOF

T or F

Larger peptides move faster through the drift space until they reach the detector while lighter, smaller, and more highly charged ions move slowly

A

F (baliktad; lighter, smaller, and more highly charged ions** move faster** through the drift space until they reach the detector, while
larger peptides move slowly.

181
Q

has revolutionized the identification of microbial species in clinical microbiology laboratories.

A

MALDI-TOF MS

182
Q

This next-generation microbial identification tool has key advantages of simplicity and robustness, making it the new best method to us

A

MALDI-TOF

183
Q

What are the criteria’s in selcting an assay?

A
  1. Sample volume
  2. Sample recovery
  3. Throughput
  4. Robustness
  5. Chemical Modification
  6. Protein Aggregation
184
Q

Criteria’s in selcting an assay

What is the required weight/amount of material to be used for each assay?

A

Sample volume

185
Q

Criteria’s in selcting an assay

Will the sample won’t be damaged/destroyed
if detected using this assay?

A

Sample recovery

186
Q

Criteria’s in selcting an assay

Amount of protein that can be
produced/detected for each sample.

A

Throughput

187
Q

Criteria’s in selcting an assay

Can the procedure can be repeated? Can the assay repeat the same procedure using the same sample and would it come up with the same result?

A

Robustness

188
Q

Criteria’s in selcting an assay

Is it sensitive and specific with this method?

A

Chemical modification

189
Q

Criteria’s in selcting an assay

Would the assay clamp or aggregate the protein?

A

Protein Aggregation

Molecular Biology Lecture (17 decks)

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