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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
Q

MCS of plc19H

multiple cloning site

A

contains LacZ
recombinant plasmids carry inserts cloned into MCS > disrupts LacZ

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26
Q

LacZ gene

A

encodes alpha peptide of beta galactosidase whose substrate is lactose

non-recombinant plc19H produce functional beta galactosidase

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27
Q

beta galactosidase detection

A

agar inclusion of X-gal> cleaved and accumulates in cells producing blue colonies

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28
Q

blue colonies

A

contain non-recombinants

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29
Q

white colonies

A

contain lacZ

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30
Q

miniprep

A

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.

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31
Q

EcoR1 restriction endonuclease in transformation

A

cuts target gene/ plasmids for matching sticky ends

gene can be inserted in either direction

reverse= nonsense

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32
Q

PCR cycle

A
  1. component mixture
  2. denaturation (94-98 C)
  3. annealing (50-68)
  4. elongation (72)
32
Q

FUP/ RUP role in PCR

forward/reverse universal primer

A

prime DNA synthesis from sequences in plc19H

33
Q

IP role

internal primer

blt101

A

primes DNA synthesis from sequence 137 bases from 5’-end on forward strand of cDNA insert
specific reaction to pblt101 recombinant plasmid

34
Q

universal primers

A

synthetic oligonucleotides complementary to specific seq in plasmid vector plC19H.

22 bases long

35
Q

blt101 internal primer

A

synthetic 22-mer complementary to sequence in cDNA insert
specific to recombinant pblt101 plasmid

36
Q

agarose gels

A

complex network of polymeric sugar molecules with a large pore size/ low frictional resistance

37
Q

DNA visualisation in gel electrophoresis

A

ethidium bromide/ Nancy520
intercalating dyes that fit between bases in DNA helix
detected via fluorescence with UV light

38
Q

2 plasmids for e.coli transformation

A

pblt101
plC19H

both have gene for ampicillin resistance/ LacZ

39
Q

DNA insertion into pblt101

A

3 primers/ 3 reactions

40
Q

PCR control reaction

A

FUP/RUP used to produce PCR product

proves reaction components are functional

41
Q

optimum temperature for RNA polymerase to replicate DNA

A

72 degrees

42
Q

restriction digestion analysis of DNA

A

via EcoR1
releases linear fragments of plasmid

43
Q

DNA migration in gel electrophoresis

A

DNA is negatively charged so will mograte towards anode

44
Q

FUP and IP function use during PCR

A

if they don’t function, suggest IP is a forward primer
therefore no product on agarose gel

45
Q

heavier band in pblt101

A

plasmid
cut pblt101 heavier than uncut pblt101

lighter band= the gene

46
Q

recombinant vs non-recombinant plasmid in PCR

A

recombinant> pblt101
non-recombinant> plc19h

47
Q

why does pblt101 have more bands than plC19H?

A

because pblt101 is recombinant > heavier is plasmid and lighter is gene
plC19H > non recombinant therefore one band as no gene inserted

48
Q

concatamer

A

above first molecular weight molecule
same sequence linked in series

reason for multiple larger bands in uncut pblt101

49
Q

what does SDS-PAGE stand for?

A

SDS-polyacrylamide gel electrophoresis

50
Q

practical 2 process

A

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
Q

blocking agent examples

A

milk powder
casein
BSA solution

52
Q

blocking agent function

A

binds to empty binding sites > primary antibodies can’t bind and give false positives

53
Q

primary antibody in western blotting

A

binds specifically to protein of interest

54
Q

secondary antibody in western blotting

A

binds primary antibody via Fc portion
conjugated to enzyme (HRP)
amplifies signal and ^sensitivity

55
Q

enzyme used in western blotting to attach to secondary antibody

A

peroxidase/ alkaline phosphatase
catalyses reaction resulting in coloured band/ chemiluminescent product

56
Q

western blotting function

A

identifies presence of specific protein

57
Q

practical 2 background

A

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
Q

method of separation of cytosolic and membrane proteins

A

centrifugation> separate fractions for examination w western blotting/ SDS

59
Q

GLUT-4 location detection

A

cytosolic/ membrane protein separation
Ig specific to GLUT4 > western blot

60
Q

SDS page method

A
  1. dilute each of 4 cell lines with loading buffer
  2. run on polyacrylamide gel
  3. separation towards anode
61
Q

4 cell lines in P2

A

323-L1 adipocyte no insulin
323-L1 adipocyte insulin
323-L1D adipocyte no insulin
323-L1D adipocyte insulin

62
Q

components of SDS loading buffer

A

SDS/ DTT/ EDTA/ Glycerol

63
Q

SDS function in loading buffer

A

denatures proteins
adds net negative charge
detergent > solubilizes protein components

64
Q

DTT function in loading buffer

A

dithiothreitol
Breaks di-sulfide bonds
maintains unfolded state

65
Q

EDTA function in loading buffer

A

ethylenediaminetetraacetic acid
cationic chelator
binds Mg/ Cl ions > insoluble salts w SDS

66
Q

Glycerol function in loading buffer

A

increases density of sample
add weight

67
Q

running buffer

A

contains tris/ glycine
even conduction of electrical current
SDS for negative protein charge

68
Q

gel transfer to nitrocellulose membrane

A
  1. submerge gel in transfer buffer
  2. sandwich and run in cold tank
  3. stirrer to spread
69
Q

western blotting

A
  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
Q

blocking buffer components

A

TBST
Casein

71
Q

tween-20

A

tween-20
detergent promoting specific antibody binding/ background noise reduction

72
Q

developing solution

A

contains substrate for secondary antibody horse radish peroxidase

73
Q

Bradford assay graph

A

x> protein conecntration\
y> absorbance at 595

74
Q

tris function in TBST

75
Q

TBST components

A

tris-HCl
tween-20
NaCl

76
Q

NaCl in TBST

A

reduces non-specific binding between antibody and aids tonicity

77
Q

HST buffer

A

Tris-HCl
NaCl

78
Q

Tris function in HST buffer

A

buffer of pH

CMB2000 (3 decks)

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