WB of lysates with FATP antibodies
Lysis Protocol
RIPA Buffer
150 mM NaCl
1% NP-40
0.5%DOC (deoxycholic acid)
0.1% SDS
50mM Tris ph 8.0
1.752 g
NaCl
2.0 ml
NP-40
1.0 g
DOC
1.0 ml
20% SDS
6.67 ml
1.5M Tris ph 8.0
fill to 200 ml with ddH2O
For Plated Cells
-rinse plate 2-3 times with PBS
-add 0.5 ml of RIPA and swirl
-using a cell scraper scrape cells
-resuspend cells using a pipet
-place into 1.0 ml microcentrifuge tube
-let sit on ice for 5 min
-spin @ high speed (12-14K) for 10 min in 4˚
-save supernatant (protein)
For Tissues
-place tissue into sterile 15 ml conical
-add about 2 ml of RIPA buffer
-polytron ~1 min on a medium setting
-spin @ 10,000 rpm, 4˚C for 10 min
-save supernatant
*you can also transfer the lysis mixture to microcentrifuge tubes to spin @12-14K.
*protease inhibitors are usually added to the lysis mixture before tissues or cells have
been disrupted.
SDS-PAGE
Prepare Samples for Loading into Gel:
-Use 5X Sample Buffer
-Prepare to load 10 µl of 1mg/ml of each sample (10µg)
-Add Sample Buffer to each tube to make 1X
-Boil the sample in heating block @ 90-95˚ for 2 min
-Remove MW marker “See Blue” from –20˚
-While boiling samples prepare gel for loading
-Dilute 10X Tris/Glycine/SDS to make 1L of 1X
-Remove gel from packet (precast novex gel 4-20% 1.5mm)
-Remove white tape and comb
-Place gel into gel box tall side facing out
-Pour in 1X Tris/Glycine/SDS to cover the gel
-Pour in the center first to ensure a good seal
-Load MW marker @10 µl / well
-Load samples at 10 µl / well
-complete electrophoresis box assembly by placing the lid on and plugging in to the
power supply
Run the gel:
-with power off
-turn dial all the way to the left
-flip the switch to constant voltage
-flip switch to high
-turn the power on and set to 110 volts
-amps should be ~50, there is a problem if it runs much higher than 50
-run for about an hour
Prepare for Transfer to Nitrocellulose Membrane:
-bring out transfer apparatus
-transfer buffer
-container to soak pieces with transfer buffer
-blotting paper ( for one gel = 2 pieces for top and 2 for bottom)
-nitrocellulose membrane
* all pieces should be sized equally to fit the gel
When gel run is complete and ready to transfer:
-disconnect electrophoresis apparatus from power supply
-soak 2 pieces of blotting paper and place in the center of the transfer apparatus
-roll over them with a broken pipette to remove air bubbles
-begin removing gel from cassette
-do not re-orient it by flipping it over
-trim away the lower portion of the gel and thelanes with a razor
-soak nitrocellulose sheet
-place atop the blotting paper
-roll to remove air
-place gel into transfer buffer
-place atop membrane
-roll to remove air
-soak 2 more pieces of blotting paper and place on top
-roll over the whole sandwich
-place the lid on the transfer device
-plug lid into bottom
-plug apparatus into power supply
Begin Transfer:
-with power off
-turn dial all the way to the left
-flip the switch to constant current
-flip switch to high
-turn the power on and set to 100 mAmps
-volts should be ~10-15
-run for 1 hour
Prepare for overnight blocking:
-prepare 5% milk in 1X PBST
-2.5g non-fat milk in 50 ml 1X PBST
-prepare containers to place the membrane into
When transfer is complete:
-remove nitrocellulose from sandwich
-place in container
-add just enough Ponceau S Solution (0.1% in 5%acetic acid) to cover the membrane
*ensure that transfer went well, there should be a band pattern for every well. If there are
any bubbles visible or abnormalities in the staining pattern record locations for later
analysis.
-swirl for 1 min
-pour off the solution
-rinse a couple of times with 1X PBST
-add blocking solution (5% milk in 1X PBST)
-incubate at 4˚ with agitation overnight
The Following Day:
-bring blot to room temp
-pour off blocking buffer
-add a little PBST, swirl, dump
Perform Primary antibody incubation
-add 10 µl of 1000X FATP antibody to 10 ml of 5% millk in PBST (dilute antibody
1/1000)
-add this to the membrane
-incubate with agitation for 1 hour @room temp
-pour off 1˚ antibody and perform PBST rinse
-add a little 1X PBST and let sit with agitation for 10 min
-do this 3 times
Perform Secondary Antibody Incubation
-add 2˚ antibody (Peroxidase Conjugated Donkey ant – rabbit)
-dilute 1/5000 in 5% milk PBST
-2 µl 2˚ antibody to 10 ml 5% milk in PBST
-incubate at room temp with agitation for 1 hour
-pour off 2˚ antibody
-perform PBST rinse 3x
Prepare Chemiluminescent Substrate Mix
-mix components as 1:1 to make enough for 2.5 ml to each membrane
-Prepare plastic wrap to place membrane onto
-get film and cassette holder
After PBST rinse, perform Chemiluminescent Rxn
-deposit 2.5 ml on to membrane
-let sit for 1 min
-lift membrane with forceps
*let membrane drain really well
-hold Kimwipes to the tip and gently wipe the back
-wrap membrane in plastic wrap
-tape to the inside of the film cassette
-expose blot to Amersham Bio-film in darkroom at the clinic
-start with 10 s, 30 s, 1 min exposures