Antigen detection on Western blotted nitrocellulose and transfection of COS7 cells with GFP plasmid

1
(No Transcript)
2
BIOE 202 Lab 8
Mar 12
Antigen detection on Western blotted
nitrocellulose and transfection of COS-7 cells
with GFP plasmid
3
Pre-Lab
Review
The reagents used in the color reaction are
alkaline phosphatase (AP or ALP), nitro blue
tetrazolium (NBT), and bromo chloro indolyl
phosphatase (BCIP) Monoclonal, not polyclonal
antibodies are created by fusing a B cell with a
tumor cell Blocking buffer is used to reduce
background binding P-16 is the polyclonal
antibody created against adipsin Advantages of
using a secondary antibody include increased
specificity and improved signal
4
Pre-Lab
Review
Cos-7 cells were isolated from monkeys Transfectio
n efficiency is decreased by antibiotics but not
by phenol red GFP transfection is used to
determine the distribution of a protein and track
movement of a protein Today, its necessary for
you to wear gloves (this experiment is BS-2) and
keep DNA on ice Cells grown in serum free media
are more fragile in trypsin
5
GFP transfection
Procedure
Practice pipetting at your bench exercise on pg
85 Aliquot serum free media into six
tubes Carefully add the proper amount of FuGENE
(just one bottle keep the cap on) to each tube,
mix and incubate in hood Add the (tiny) amount of
DNA to each tube, mix and incubate in hood
leave tips in waste container Add solution to
6-well plate of COS-7 cells use the automatic
pipettor with a pasteur pipet
6
Antigen detection
Procedure
Soak and rock your nitrocellulose in blocking
buffer (5 nonfat dry milk in TBST) Add 1 nonfat
dry milk in TBST and agitate Add primary
antibody, agitate for 60 min Rinse and agitate in
1 solution 3x Add secondary antibody with 1
solution and agitate for 50 min Rinse again Add
NBT BCIP, develop in dark for 5-10 min Dry in
air
7
Reminders
While youre waiting for antigen detection
complete the pipetting practice and watch Growing
Spare Parts For GFP transfection wear gloves at
all times with COS-7 cells and keep DNA on
ice You will return on Thursday to observe your
cells If you would like your lab report regraded,
hand it in to me before Spring Break. Remember
to hand in your original copy as well.