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CMB2000 > practicals > Flashcards

practicals Flashcards

1
Q

stages of capture ELISA

A
  1. Capturing Ig absorbed onto solid phase
  2. Ag added (transporter protein)
  3. labelled detection Ig added > binding in presence of Ag
  4. substrate added w HRP label
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2
Q

HRP label

A

catalyses oxidation of substrate for color change
Ag conc proportional to color produced

horse radish peroxidase

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3
Q

characteristic of capture and detector antibody

A

cross-reactive

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4
Q

total protein concentration determination in ELISA

A

Colorimetric assay

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5
Q

determination of novel protein percentage of total protein in test sample

A

use of 2 assays > capture ELISA/ colorimetric assay

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6
Q

what does ELISA stand for

A

Enzyme-linked immunosorbent assay

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7
Q

ELISA error factors

A

insufficient plate washing
oversaturation of wells with conjugate
contamination
incubation timing
standard curve range too high

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8
Q

ELISA advantages

A

high efficiency
simultaneous analysis w/o pre-sample treatment
safe/ eco-friendly

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9
Q

ELISA disadvantages

A

antibody instability
refridgerated storage and transport of antibody

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10
Q

tween-20 in ELISA

A

non-ionic detergent decreasing asorption of non-specific Ig to ELISA wells. Block vacant binding sites by *H bonds/ Van der Waals. *

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11
Q

Use of calf serum in ELISA

A

contains albumin
blocks non-specific binding

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12
Q

need for 2 antibodies in ELISA

A

primary antibody is specific/ facilitates immbolisation of antigen
secondary antibody contains enzyme and has wider range of targets> facilitates Ag detection

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13
Q

5 classes of antibodies

A

IgA/ IgE/ IgD/ IgG/ IgM

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14
Q

IgA

A

protects against pathogens

Found in mucous, saliva, tears, and breast milk.

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15
Q

IgD

A

activates basophils and mast cells

part of BCR

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16
Q

IgE

A

role in allergic reactions

protects against parasitic worms

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17
Q

IgG

A

can cross placenta into foetus
secreted by plasma cells into blood

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18
Q

IgM

A

early stages of immunity

secreted into blood / surface of B cell

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19
Q

what does practical 1 entail?

A

plasmid isolation, PCR and electrophoretic analysis of restriction digested DNA

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20
Q

recombinant plasmid in practical 1

A

cDNA sequence cloned into spec EcoR1 site
blt101 cDNA insert

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21
Q

miniprep

A

isolation of plasmid DNA from small-scale bacterial culture

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22
Q

how do you make E.coli cell competent?

A

CaCl2/ low temperature treatment of cells

making cell wall permeable to DNA> plasmid uptake

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23
Q

transformation

frequency in E.Coli cells

A

plasmid uptake
1 in 100 cells maximum

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24
Q

plC19H

A

non-recombinant/ no novel gene inserted in lacz
carries gene enabling ampicillin resistance

enables detection of transformants in ampicillin medium

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25
MCS of plc19H | multiple cloning site
contains LacZ recombinant plasmids carry inserts cloned into MCS > disrupts LacZ
26
LacZ gene
encodes alpha peptide of beta galactosidase whose substrate is lactose | non-recombinant plc19H produce functional beta galactosidase
27
beta galactosidase detection
agar inclusion of X-gal> cleaved and accumulates in cells producing blue colonies
28
blue colonies
contain non-recombinants
29
white colonies
contain lacZ
30
miniprep
solutions (detergent/ alkali mixture) lyse bacterial cells, denature/ solubilise cellular components/ allow RNAase to degrade RNA. Plasmid DNA trapped on matrix, contaminants washed away. plasmid DNA released/ eluted from miniprep column.
31
EcoR1 restriction endonuclease in transformation
cuts target gene/ plasmids for matching sticky ends | gene can be inserted in either direction ## Footnote reverse= nonsense
32
PCR cycle
1. component mixture 2. denaturation (94-98 C) 3. annealing (50-68) 4. elongation (72)
32
FUP/ RUP role in PCR | forward/reverse universal primer
prime DNA synthesis from sequences in plc19H
33
IP role | internal primer ## Footnote blt101
primes DNA synthesis from sequence 137 bases from 5'-end on forward strand of cDNA insert specific reaction to pblt101 recombinant plasmid
34
universal primers
synthetic oligonucleotides complementary to specific seq in plasmid vector plC19H. | 22 bases long
35
blt101 internal primer
synthetic 22-mer complementary to sequence in cDNA insert specific to recombinant pblt101 plasmid
36
agarose gels
complex network of polymeric sugar molecules with a large pore size/ low frictional resistance
37
DNA visualisation in gel electrophoresis
ethidium bromide/ Nancy520 intercalating dyes that fit between bases in DNA helix detected via fluorescence with UV light
38
2 plasmids for e.coli transformation
pblt101 plC19H | both have gene for ampicillin resistance/ LacZ
39
DNA insertion into pblt101
3 primers/ 3 reactions
40
PCR control reaction
FUP/RUP used to produce PCR product | proves reaction components are functional
41
optimum temperature for RNA polymerase to replicate DNA
72 degrees
42
restriction digestion analysis of DNA
via EcoR1 releases linear fragments of plasmid
43
DNA migration in gel electrophoresis
DNA is negatively charged so will mograte towards anode
44
FUP and IP function use during PCR
if they don't function, suggest IP is a forward primer therefore no product on agarose gel
45
heavier band in pblt101
plasmid cut pblt101 heavier than uncut pblt101 | lighter band= the gene
46
recombinant vs non-recombinant plasmid in PCR
recombinant> pblt101 non-recombinant> plc19h
47
why does pblt101 have more bands than plC19H?
because pblt101 is recombinant > heavier is plasmid and lighter is gene plC19H > non recombinant therefore one band as no gene inserted
48
concatamer
above first molecular weight molecule same sequence linked in series | reason for multiple larger bands in uncut pblt101
49
what does SDS-PAGE stand for?
SDS-polyacrylamide gel electrophoresis
50
practical 2 process
separation via SDS-PAGE gel electrophoresis and transfer onto nitrocellulose membrane (high binding affin for proteins) blocking agent addition incubation w primary antibody then secondary > enzyme conjugation
51
blocking agent examples
milk powder casein BSA solution
52
blocking agent function
binds to empty binding sites > primary antibodies can't bind and give false positives
53
primary antibody in western blotting
binds specifically to protein of interest
54
secondary antibody in western blotting
binds primary antibody via Fc portion conjugated to enzyme (HRP) amplifies signal and ^sensitivity
55
enzyme used in western blotting to attach to secondary antibody
peroxidase/ alkaline phosphatase catalyses reaction resulting in coloured band/ chemiluminescent product
56
western blotting function
identifies presence of specific protein
57
practical 2 background
adipocytes given insulin PL3K/ PKB cause GLUT4 translocation from cytosol to PM cell lines produced w disrupted aPKC via CRISPR/Cas9 > GLUT4 translocation proves CRISPR success
58
method of separation of cytosolic and membrane proteins
centrifugation> separate fractions for examination w western blotting/ SDS
59
GLUT-4 location detection
cytosolic/ membrane protein separation Ig specific to GLUT4 > western blot
60
SDS page method
1. dilute each of 4 cell lines with loading buffer 2. run on polyacrylamide gel 3. separation towards anode
61
4 cell lines in P2
323-L1 adipocyte no insulin 323-L1 adipocyte insulin 323-L1D adipocyte no insulin 323-L1D adipocyte insulin
62
components of SDS loading buffer
SDS/ DTT/ EDTA/ Glycerol
63
SDS function in loading buffer
denatures proteins adds net negative charge detergent > solubilizes protein components
64
DTT function in loading buffer
dithiothreitol Breaks di-sulfide bonds maintains unfolded state
65
EDTA function in loading buffer
ethylenediaminetetraacetic acid cationic chelator binds Mg/ Cl ions > insoluble salts w SDS
66
Glycerol function in loading buffer
increases density of sample add weight
67
running buffer
contains tris/ glycine even conduction of electrical current SDS for negative protein charge
68
gel transfer to nitrocellulose membrane
1. submerge gel in transfer buffer 2. sandwich and run in cold tank 3. stirrer to spread
69
western blotting
1. membrane into blocking buffer 2. replace w primary antibody solution 3. wash and replace with secondary antibody solution 4. HST buffer and developing solution 5. record on gel-doc
70
blocking buffer components
TBST Casein
71
tween-20
tween-20 detergent promoting specific antibody binding/ background noise reduction
72
developing solution
contains substrate for secondary antibody horse radish peroxidase
73
Bradford assay graph
x> protein conecntration\ y> absorbance at 595
74
tris function in TBST
buffer
75
TBST components
tris-HCl tween-20 NaCl
76
NaCl in TBST
reduces non-specific binding between antibody and aids tonicity
77
HST buffer
Tris-HCl NaCl
78
Tris function in HST buffer
buffer of pH

CMB2000 (3 decks)

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