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bioanalytical > protein 2 > Flashcards

protein 2 Flashcards

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1
Q

What is protein electrophoresis?

A

separation technique based on the
migration of charged proteins in an
electric field

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2
Q

What are some characteristics of protein electrophoresis?

A

allows the estimation of
protein size and charges and
the purity of a protein sample

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3
Q

What is the term used to define the movement of a molecule in an electric field?

A

mobility (u)

which equals to v/E, the velocity per unit of electric field (E)

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4
Q

How to calculate mobility?

A

mobility = v/E = q/f

where
q: net charge
f: frictional coefficient

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5
Q

Vertical slab gel electrophoresis is the most common analysis technique in biomedical laboratories.

True or false

A

True

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6
Q

What kind of gel electrophoresis is a method of choice for protein analysis?

A

Polyacrylamide gel electrophoresis

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7
Q

How is polyacarylamide gel made?

A

polymerization of acrylamide and crosslinking N,N’-
methylenebisacrylamide

–> diff porous sizes depending on % of polyacrylamide

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8
Q

What are the steps in vertical slab polyacrylamide gel electrophoresis?

A
  1. Protein samples applied to wells on the top
  2. Electric field applied between top and bottom of gel (anode at the bottom).
  3. proteins migrate from top to bottom
  4. protein bands visualized with diff staining
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9
Q

What property of proteins is SDS-PAGE based on?

A

sizes

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10
Q

What property of proteins is native PAGE based on?

A

physical chemical properties

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11
Q

What property of proteins is isoelectric focusing (IEF) based on?

A

isoelectric point

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12
Q

Name 3 types of gel electrophoresis techniques for separation of proteins.

A
  1. SDS-PAGE
  2. native PAGE
  3. IEF
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13
Q

Name a type of staining that could be used in GE to visualise different bands of proteins

A

Commassie Blue

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14
Q

In GE, the higher the acrylamide conc (%), the _______ the linear range of separation

A

lower

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15
Q

Why is it named ‘Native’ PAGE?

A

proteins samples kept in native state

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16
Q

In Native Page, what factors may protein migration depend on?

A

size, shapes and charges of proteins and
the porous density of the polyacrylamide gel

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17
Q

Native Page is used to study ___________________________________.

A

protein polymerization, protein interaction and the uniform conformation of a purified protein

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18
Q

In Native Page, what rxn may be carried out to confirm the activity?

A

In-gel enzymatic rxn (zymography)

–> using different enzymatic rxns to to identify enzymes such as lactate dehydrogenase

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19
Q

Describe the structure of lactate dehydrogenase (LDH).

A
  • tetrametric proteins
  • 2 types of subunits, M (muscle) and H (heart) –> M found in muscle, and H found in heart
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20
Q

How is zymography in native PAGE carried out?

A
  1. gel contains substrate
  2. sample contains enzyme and digests substrate in gel
  3. substrate stained
  4. clear zone indicate activity
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21
Q

What does ‘SDS’ in SDS PAGE stand for?

A

sodium dodecyl sulfate

–> an anionic detergent that unfolds proteins and provides them with negative charges.

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22
Q

In SDS PAGE, what is bound SDS proportional to?

A

proportional to the length of the
polypeptide chain (~1.4 g SDS/g
protein)

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23
Q

What is protein separation in SDS PAGE based on? How?

A

molecular mass

velocity is inverse linear function of logarithm of molecular mass

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24
Q

How could molecular mass of protein be estimated thru SDS PAGE?

A

Proteins of known molecular mass
can be used to establish a calibration curve (a descending line)

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25
What is to be done to proteins before denaturing them with SDS in SDS PAGE?
reduce disulphide bonds with 2-mercaptoethanol
26
In SDS PAGE, proteins are unfolded to a linear structure with a negative charge proportional to the _______________________.
polypeptide chain length
27
What could be one function of SDS PAGE?
Check the purify of protein sequential steps of chromatographic purification
28
What are some dyes that are used in SDS PAGE?
1. organic dyes such as Coomassie blue 2. silver stain
29
Which is more sensitive? Coomassie blue or silver stain?
silver stain: 10-100X more sensitive
30
What is the principle of isoelectric focusing (IEF)?
1. polyacrylamide gel with generated pH gradient in electric field 2. Mixtures of ampholytes used to generate pH gradient 3. proteins move until reach net 0 charges at their PI
31
In IEF (isoelectric focusing), what is used to generate the pH gradient?
Mixtures of ampholytes, small amphoteric molecules with high buffering capacity near their pI,
32
What is the principle of 2D-PAGE?
Proteins are separated first by isoelectric focusing, then by an SDS-PAGE --> protein separation baed on both the isoelectric point (pI) and size of proteins
33
What is DIGE and what are the steps involved with it?
Differential In-Gel Electrophoresis 1. Proteins from each sample fluorescently labeled with distinct dyes 2. A pooled internal standard (representing all samples) labeled with a third dye 3. protein separation using 2D PAGE 4. fluorescence detection
34
What are some fluorescent dyes used in DIGE?
Cy2, Cy3 and Cy5
35
What is western blotting?
a method that uses antibody to detect the presence of a particular protein in a sample (blood, tissues etc)
36
What are the steps in western blotting?
1. SDS PAGE 2. proteins electrically transferred to blotting membrane (nitrocellulose, PVDF) 3. membrane incubated with primary antibody specific to protein of interest 4. second antibody linked with enzyme (HRP) added 5. second antibody bind to primary antibody 6. substrate added to generate fluorescence, luminescence or color precipitate
37
What is the direction of transfer of proteins to blotting membrane?
Cathode (-) to Anode (+)
38
What is the function of IgG?
The most abundant in B cells and serum
39
What is the function of IgA?
Secreted in saliva, mucus and breast milk as neutralizing antibodies to present pathogen infection
40
What is the function of IgM?
As pentamers joined by linker. First to be produced by B-cells against a new infection
41
What is the function of IgD?
Present on the surface of B cells
42
What is the function of IgE?
Present in mast cells associated with allergy
43
What is Immunoprecipitation (IP)?
precipitation method to isolate a specific protein antigen from a mixture, using the antigen-antibody interaction
44
What are the steps involved in immunoprecipitation (IP)?
1. primary antibody added to antigen 2. secondary antibody or protein A bound to agarose beads and magnetic beads added to mixture 3. washing 4. elution 5. SDS-PAGE or western blotting
45
In IP, how are isolated proteins usually analyzed?
SDS-PAGE, Western blotting, Mass spectrometry
46
Describe the steps involved in Pull down assay for the study of protein-protein interactions?
1. immobilize fusion tagged 'bait' from lysate using affinity ligand attached to agarose bead 2. wash away unbound protein 3. bind 'prey' proteins to immobilized 'bait' protein 4. wash away unbound protein 5. elute protein-protein interaction complex 6. analyze interaction by SDS PAGE
47
What is a 'bait' protein?
protein tagged with a fusion tag (e.g., GST, poly-Histidine, biotin). captured by affinity ligand on agarose beads
48
What is the full form of ELISA?
Enzyme Linked ImmunoSorbent Assay
49
What are the steps involved in ELISA?
1. Antigen of interest absorbed on to plastic surface 2. Antigen recognized by antibody 3. antibody recognized by 2nd antibody linked with enzyme 4. substrate react with enzyme to give color 5. color measured
50
In what conc range can ELISA measure antigen?
pg-ng/mL
51
Name some enzymes that are usually linked to 2nd antibody in ELISA.
horse radish peroxidase(HRP) alkaline phosphatase (AP)
52
What are the different types of ELISA?
1. direct elisa 2. indirect elisa 3. sandwich elisa 4. competitive elisa
53
What is the most common format of ELISA?
indirect ELISA
54
What is competitive ELISA used for?
Quantitative detection of competitive antigen
55
What is sandwich ELISA used for?
to detect antigen
56
What is sandwich ELISA?
relies on the binding of the target antigen between two layers of antibodies: a capture antibody and a detection antibody. (detection antibody could be one or could be a combo of primary and secondary antibody)
57
What are the 2 commonly used linked-enzymes in ELISA?
1. horseradish peroxide (HRP) 2.Alkaline phosphatase (AP)
58
What is the chromogenic substrate of Horseradish peroxidase?
Tetramethylbenzidine (TMB) --> The absorbance of the color at 450 nm --> chemifluorescence
59
What is the chromogenic substrate of Alkaline phosphatase (AP)?
p-Nitrophenyl Phosphate (pNPP) --> Yellow product at 405 nm
60
What are the different formats of signal detection of ELISA?
1. bioluminiscence (eg luciferase- conjugated Ab) 2. chemifluorescence 3. autoradiography 4. immunogold
61
What are the steps involved with chemifluorescence-based immuno-magnetic bead assay?
1. synthetic peptide preparation, peptides biotinylated 2. biotinylated peptide mix with streptavidin beads 3. addition of Patient Serum --> antibodies in serum bind to biotinylated peptide-bead complex 4. Wash + add secondary antibody 5. add substrate 6. measure chemifluorescence
62
Explain the principle of lateral flow immunodiffusion (immuno- chromatography assay) for detection of covid
1. colloid gold nanoparticle antibody for covid antigen on conjugate pad 2. move on nitrocellulose paper support 3. test line contain another antibody for antigen --> sandwich ELISA 4. control line contain antibody for for the colloid gold nanoparticle antibody
63
What is hCG?
chorionic gonadotropin (hCG)
64
What is the main difference between Colloidal gold-immunochromatographic assay (GICA) and Lateral flow immunodiffusion (immuno- chromatography assay)?
GICA detects antibodies produced by covid patients and tests plasma and sera Lateral flow immunodiffusion detects covid antigens in patients and tests saliva mostly
65
Explain the principle of colloidal gold-immunochromatographic assay (GICA) for the rapid detection of SARS-CoV-2 antibodies.
1. release pad contains colloidal gold particles conjugated to SARS-CoV-2 antigens --> antibodies in test sample bind to it 2. move through nitrocellulose membrane 3. T1: anti-human IgM antibodies that capture IgM antibodies bound to the gold-antigen complexes. 4. T2: anti-human IgG antibodies that capture IgG antibodies bound to the gold-antigen complexes 5. C: immobilised anti-rabbit IgG antibodies, which bind to gold-labeled rabbit IgG (internal control)
66
What are the pros of colloidal gold-immunochromatographic assay (GICA) for the rapid detection of SARS-CoV-2 antibodies?
 Precision  Limit of detection  Accuracy  Specificity
67
What is the full form of GICA?
Colloidal gold-immunochromatographic assay
68
Protein levels and their post- translational modification (PTM) change constantly in their dynamic states, reflecting their normal physiological function, any abnormal changes may indicate disease progression.
True
69
Proteins translocate in different cellular organelles in response to stimuli
True
70
Why is system-based proteomic study preferred?
allows us to identify new signalling pathways and to design new drugs.
71
What are the 2 predominant ionisation approached in MS?
1. Electrospray Ionization (ES) 2. Matrix Assisted Laser Desorption Ionization(MALDI)
72
What is a common matrix in MALDI-TOF MS/MS?
2,5-dihydroxybenzoic acid (DHB)
73
What are the 2 different methods in quantitative mass spectrometry?
1. Chemical labelling: iTRAQ and TMT 2. metabolic labelling: SILAC
74
How does iTRAQ and TMT work?
1. diff experimental groups 2. protein digestion 3. peptide labelling 4. mix peptides 5. quantification by MS2
75
How does SILAC, a metabolic labelling technique in quantitative MS work?
1. control grown in light medium and experimental group grown in heavy medium 2. mix lysates 1:1 3. protein digestion 4. quantification by MS1
76
What is light and heavy medium in SILAC?
light medium: Contain naturally occurring isotopes (e.g., ¹²C, ¹⁴N). heavy medium: Contain stable isotopes (e.g., ¹³C, ¹⁵N) arginine and lysine are typically used as they appear in all tryptic peptides
77
Name some common PTMS
phosphorylation acetylation N-linked glycosylation amidation
78
Name some AA residues that can undergo phosphorylation
serine, threonine, tyrosine, histidine
79
Describe Alzheimer's disease
progressive, neurodegenerative disease --> abnormal clumps (amyloid plaques) and tangled bundles of fibers (neurofibrillary tangles) composed of misplaced proteins called Tau in the brain --> deficit in cholinergic transmission in the affected brain area, causing cognitive dysfunction
80
Which is the most common type of ELISA?
indirect ELISA
81
What is the difference between direct and indirect ELISA?
direct: 1 antibody (enzyme linked) indirect: 2 antibodies
82

bioanalytical (10 decks)

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