immuno lab pt 2

what does western blotting tell us

protein size

antibody specificity

how are proteins sorted by size

SDS-PAGE

• electrophoresis

what are the proteins of interest in the fish samples

actin

myosin light chain 1/2

why was a bradford preformed on the fish samples

to determine inital protein concentration

• we want to load the same amount of protein into the SDS PAGE so we need to 1st know how much protein there is in the sample

how much protein was loaded into the SDS-PAGE

50mg

how did you determine how much fish sample to load into the gel

1. bradford to determine starting protein concentration

   1. standard curve interpolation

2. determine the actual concentration

   1. standard curve interpolation * dilution factor

3. 50mg / concentration of sample (mg/ul)

   1. if above 10ul then load that amount

   2. if under 10ul do a dilution to the sample

4. if diluted: determine new load value

   1. 50mg / new concentration

why do we want to standardize how much protein is loaded into the gel

when we qualitative comparison of bands results it accurately reflects the true concentration of each protein in the sample

• fish 1 has a lot of mysoin light chain 1 b/c it naturally does not b/c it has more protein when it was loaded

what is laemmli made of that lyses cells and dentautes proteins

2% sds

DTT

SDS

lyses cells

coats protein in (-) charges

DTT

breaks disulfide bonds between amino acids → destorys 2nd structure

how can we denature a protein

SDS

DTT

heat

how do we image the gel

UV light in the biorad imager

how are proteins run in a gel

from (-) to (+)

set up of gel blotting sandwhich

black cassette

fiber pad

filter paper

gel

membrane

filter paper

fiber pad

red cassete

what is the reversiable dye used to check that the gel bands transfered to the membranes

amino black

why must we soak the gel in blotting buffer before we do the sandwhich

to pre shrink the gel

• gel will shrink during the sandwhich → distorts bands

• to prevent this we pre shrink the gel

why do we block the membrane prior to immunodetection

to prevent non specific binding of the antibodies

myosin light chain ½ control kd

myosin light chain 1= ~21

myosin light chain 2= ~19

how can you be sure that a 21kd band is myosin light chain

immunodetetcion

what is a miccroarray

measures the expression levels of genes or proteins

how do you get from isolated mrna to dna for a miccroarray

reverse transcriptase

• mrna → cdna

red in miccroarray

cancer genes are upregulated

normal genes are downregulated

yellow in miccroarray

cancerous and normal genes are expressed equally

green in miccroarray

normal genes are upregulated

cancer is downregulated

how can we verify a dna miccroarray

protein miccroarray

why do some protein arrays not correspond to the dna miccroarrays

translations modifications in the protein

• phosphorolation

• glycosylation

protein miccroarray

1. isolate protein

2. label (red or green)

3. bind to miccroarray with antibodies

4. determine profile

whats the best way to view miccroarray data

preform a log2 transformation

• make patterns in data easier to see

log 2 > 0

• meaning for miccroarray

gene is pro-cancerous

log 2 < 0

• meaning for miccroarray

genes is expresses in normal cells

• repressed in cancer

log 2 = 1

• meaning for miccroarray

genes are equally express in cancer/normal cells

cluster analysis

helps identify subtypes of cancer and effective treatments

what do you need to do PCR

taq pol

DNTPs

magnesium ions

buffer system

sample DNA

process of PCR

melt—- denature DNA

anneal—- attatch denatured helix to new primers

extend —- create new DNA stands

how many cycles do we typically do in PCR

30-40

Tm

temp at which 50% of the primer will anneal to the DNA template

endpoint PCR

does patient have HIV?

do gel elecrophoresis after PCR is completed to see if fragments were isolated

QPCR

tells how much HIV paitent has

uses a reporter molecule that when incorperated into the DNA it floreses. This can tell us how much DNA is present at all times during the PCR

• more floressence = more DNA present

threshold cycle (Ct)

when the floursence of a sample reaches a set threshold level

• more starting dna will get to the Ct faster

melt curve analysis

tells us if we isolate dna from 1 source.. take the negative first derativite to make graph easier to interpret

• 1 peak = 1 source of dna

• 2+ peaks= 2+ sources of dna

how long is a typicaly PCR primer

15-30 nt

What is in laemilli that could cause interferance with bradford

• how do we fix this

SDS

dilute the fish protein extract that has laemilli/SDS to a concentration less than 2%

a large protein will move — through an SDS gel compared to a small protein

slower

why do we transfer the fish protein onto a membrane for immunodetection

antibodies cannot penetrate the SDS gel

what in the transfer buffer / blotting buffer causes the gel to shrink

methanol

what happens if there are air bubbles between the gel and the membrane

proteins will not transfer properly

why did we stain the membrane with amido black

to mark down the locations of the ladder and to ensure we transfered proteins onto the membrane

how much liquid do you load into each lane of the SDS-PAGE

standards: 20ul

samples: 50ug/ bradford (diluted) concentrations

for immunospecificity what is the type of primary/secondary/subtrate used

primary= monoclonal antibody

secondary= polycolonal antibody

substrate= HRP

for immunospecificty what was the concentration of the primary and secondary antibodies

primary= 1:200

secondary: 1:800

can antibodies be reused

yes

so long as sodium azide is present in the solution to prevent bacterial growth

how do we get a log2 value

In (ratio of cancer to normal gene expression) / In 2

PCR curve

1. exponential= 2x replication each round.. plenty of nutrients/subtratre

2. linear= not quite 2x repli each round… nutrients decrease

3. plateau= no more replication.. no more nutrients