western blotting and brdu staining
TRANSCRIPT
WESTERN BLOTTING WESTERN BLOTTING AND BAND BRDRDU STAININGU STAINING
CHRISTIAN LAPITANCHRISTIAN LAPITANMILLIKAN HIGH SCHOOL – LONG BEACH
AMRITA AHLUWALIA, PH.D.AMRITA AHLUWALIA, PH.D.GASTROINTESTINAL (GI) RESEARCH
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
Methods learned so far
Cell CultureProtein isolationBradford’s assay for
determining protein concentration
SDS-PAGEWestern TransferImmunoblottingBrdU assay
Cells used:• HCT116 (colon cancer
cells) • NCM460 (normal
colonic mucosal cells)
Western BlotWestern Blot
Protein detection Gel Electrophoresis
Gel matrix: pore size is inversely proportional to gel concentration
Protein samples (-) in the wells move to the positive anode (+)
The size and weight of each protein determines how far it moves Top
Bottom
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
Western BlotWestern Blot
Membrane Transfer
Antibody Detection
Resulting Image
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
SecondarySecondary
PrimaryPrimary
Western BlotWestern Blot
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
NC
M 4
60
HC
T116
9
VEGF (21 kD)β-actin (42 kD)
VEG
F pr
otei
n(r
elat
ive
dens
ity)
Cells used: •HCT116 (colon cancer cells) •NCM460 (normal colonic mucosal cells
Increased VEGF in CRC cells
BrdU StainingBrdU Staining
This assay is used to quantify cell proliferation Cells used: HCT116 (colon cancer cells) and NCM460
(normal colonic mucosal cells) Cells were grown on coverslip, and serum starved overnight Cells were treated with BrdU to allow for BrdU
incorporation into DNA Cells were fixed and DNA was denatured Immunostaining was performed using BrdU antibody
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
BrdU StainingBrdU Staining
Amrita Ahluwalia, Ph.D.Gastrointestinal(GI) Research
Normal cells
CRC cells
Increased Cell Proliferation in CRC cells
P < 0.01* *
Normal cells Colon cancer cells
Cel
l pro
lifer
atio
nC
ell p
rolif
erat
ion
(% B
rdU
sta
ined
cel
ls)
(% B
rdU
sta
ined
cel
ls)