‫בס"ד‬
Lysate Preparation:
1. Prepare the lysis buffer according to the following table:
for
350
ml
Lysis
for
for
Buffer 10ml
50ml
Tris pH
8 ,1M
0.5
17.5
2.5
NP40
0.1
3.5
0.5
NaCl
5M
0.3
10.5
1.5
MgCl2
1M
0.01 0.35
0.05
Glycerol
1
35
5
DDW 8.09 283.15 40.45
10
350
50
2.
3.
4.
5.
6.
7.
8.
9.
Store the lysis buffer at 4 C.
Prepare PI X 7 solution (PI tablet at 1.5 ml DDW). Store the PI
solution at -20 C. The PI X 7 solution is stable for several months at
-20 C. Add PI solution to the lysis buffer. The final concentration of
the PI need to be X 1.
Scratch cells or trypsinize cells for 5 minutes.
If you trypsinize cells, neutralize trypsin with 1:1 ratio medium.
Centrifuge cells 5 minutes at 1.5 rpm.
Aspirate supernatant.
Add DTT to lysis buffer containing PI (1µl 1M DTT : 500µl lysis
buffer).
Lyse cells with ice-cold lysis buffer. Pipette until cells thoroughly
mixed.
Put cells on ice for 1/2 hour.
Centrifuge cells at max speed (13,000 rpm) for 20 minutes at 4 C.
10. Transfer supernatent to clean eppendorf.
11. Prepare bradford reagent (1ml Bradford: 4 ml DDW).
12. Check protein concentration (use triplicates):
by spectrophotometer - 1ml bradford : 1µl protein lysate.
by ELISA – 250 µl bradford: 1 µl protein lysate.
13. Store lysate at -80 until needed.
SDS-PAGE:
1. Choose the relevant % Acrylamide gel according to relevant
protein's size. You can use the following table:
2. Prepare the relevant Acrylamide gel according to following table:
Resolving gel:
S
t
a
c
k
i
n
g
g
e
l
:
3.
4.
5.
6.
Important:
A. Add the TEMED at the chemical hood!!!
B. Add the APS last. Load the gel immediately after adding the
APS.
Prepare the resolving gel first. Add DDW above the resolving gel.
After the resolving gel was solidified, throw out the DDW and add
the stacking gel above the resolving gel.
Dilute the protein samples with sample buffer(SB)*. The final
concentration of the SB need to be X1.
Boil the protein samples for 5 minutes.
After the stacking gel was solidified, load the protein samples at
the Acrylamide gel. Don't forget to load protein marker (3-6 µl) at
one of the wells (usually the well at one of the edges).
7. At one "running device" you can use 2 gels and you need 1 liter of
running buffer X 1**. You can reuse the running buffer 3-4 times.
8. Usually, start the running process at 180 V. Stop the running
process when the separation at the relevant protein's size is clear.
*protocol for Sample buffer x 5 (50ml):
Tris HCl 1M pH6.8
SDS (danger to breath)
15.5ml
5gr
Glycerol (liquid at room temperature)
25ml
Bromo-phenol blue
0.016gr (16mg)
DTT (at chemical hood)
3.85gr
H2O
4ml
** protocol for running buffer x 10:
Add 100 ml SDS 10%, 30.3 gr Tris and 144.1 gr Glycine. Adjust to final
volume of 1 liter with DDW.
TRANSFER:
1. Transfer buffer X 10: Add 288.2 gr Glycine and 60.4 gr Tris to 1.6
liter DDW. Mix with gentle hot and adjust to final volume of 2
liter.
2. Prepare transfer buffer X 1 (700 ml DDW, 200 ml MeOH, 100 ml
transfer X 10).
3. Prepare the "transfer sandwich":
At "wet" transfer, the gel is under the Nitrocellulose/PVDF
membrane. At "semi-dry" transfer, the gel is above the
Nitrocellulose/PVDF membrane.
4. According to the protein size, decide the duration of the transfer
process and the current strength. For example: The duration of
the wet transfer of SIRT1 (110KDa) is about 2 hours, and the
current's strength is 250 mA. You can also do the transfer for 1
hour at 100V. The duration of the semi-dry transfer of SIRT1
(110KDa) is about 1 hour, and the voltage's strength is 12.5 V.
BLOCKING:
For blocking, use 5% skim milk solution (5 gr skim milk at 100 ml
TBSTX1) or 5% BSA solution (5 gr BSA at 100 ml TBSTX1). Do
blocking at room temperature for 1 hour or at 4 C for over-night.
Primary antibody:
Dilute the primary antibody at 5% skim milk solution or at 5% BSA
solution (usually 1µl primary antibody : 1000 µl skim milk/BSA solution).
1. Incubate the membrane with the dilute solution of the primary
antibody under agitation. Usually the agitation is for over-night
at 4 C.
2. Wash the membrane with TBST X 1 for 3 times. Every wash is
for 10 minutes.
Secondary antibody:
1. Choose the relevant secondary antibody according to animal
source of the primary antibody. The horseradish peroxidase
(HRP) enzyme is conjugated to the secondary antibody in order
to detect the proteins.
2. Dilute the secondary antibody at 5% skim milk solution or at
5% BSA solution (usually 1µl primary antibody : 10,000 µl skim
milk/BSA solution).
3. Incubate the membrane with the dilute solution of the
secondary antibody under agitation. Usually the agitation is for
1 hour at room temperature.
3. Wash the membrane with TBST X 1 for 3 times. Every wash is
for 10 minutes.
Chemiluminescent detection:
The Enhanced chemiluminescent (ECL) substrate for horseradish
peroxidase (HRP) enzyme provides detection of the proteins.
1. Prepare detection mix (1ml Luminol/Enhancer : 1 ml Stable
peroxidase buffer).
2. Incubate blot in the detection mix for several seconds. During
the incubation the substrate will luminesce.
3. Detect the light by photographic film or by camera.
4. Analyze the image by densitometry (which quantifies the
results in terms of optical density).
Stripping:
1. Incubate the membrane at TBST X 1 for 3/4 hour at room
temperature.
2. Wash the membrane 3 times with DDW. Each Wash is for 5
minutes.
3. Incubate the membrane at re-blot solution X 1 (dilute re-blot X 10
with DDW) for 1 hour at room temperature.
4. Wash the membrane 3 times with TBST X 1. Each Wash is for 10
minutes.
5. Ready for blocking stage.
Important:
Check that the stripping process succeeded. If you strip primary
antibody, incubate the membrane with secondary antibody and
ECL. If you strip secondary antibody, incubate the membrane with
ECL. In the both cases no signal should appear.
Tissue culture
Media preparation
For each bottle of medium add:
- Serum ( 10%)
- Antibiotics ( 1%)
- L-glutamine (1%)
Note that different media may need different supplements (warmly
recommend to take a look at the ATCC website).
A word about contamination: routinely check for mycoplasma!
Freezing and Thawing cells
Freezing
Trypsinize the cells and spin them down (~1500 RPM, 5')
Aspirate and resuspend the cells (in freezing vials) in freezing medium
(10% DMSO in serum/ medium)- ~ 1 ml.
Put the tube in an ice bucket .
Insulation is best accomplished with cotton.
Then put the box in the -80°C freezer overnight, for slow cooling.
Don't forget to keep a vial in the laboratory stock!
Thawing
Cells should be thawed rapidly and then diluted slowly into warm
growth medium.
Thaw the vial in water bath at 37 degree, and then add it to 10 cm plate
containing warm medium.
Transfection methods (optimized for 6-well; scaling up & down is
required for optimal results)
Calcium Phosphate
Solution A: 67.5 µl of DDW+ DNA (desired amount) + 7.5 µl of CaCl2
Sterile 2.5 M.
Solution B: 86 µl of HBS*2 (hepes buffered saline).
Add Solution A to Solution B drop-wise while bubbling.
Let stand at room temp for ~ 20 min and then apply the mix on cells.
Note: the pH of HBS*2 should be 7.05 (as deviation from this will
compromise the transfection efficiency).
Metafectene
In a 6 well tissue culture plate, seed 1-5 x 105 cells in 2 ml of full growth
medium so that they will be 90-85% confluent at the time of
transfection in 2 ml of suitable fresh complete medium.
The DNA stock solution and transfection reagent should be at room
temperature. Agitate the stock solutions gently before use.
Prepare the following solutions:
Solution A: 0.4 – 5.0 µg DNA in 100 µl serum and antibiotic free medium
or PBS
Solution B: 2.0–35 µl of METAFECTENE™ (I found that twice the amount
of DNA yields best results) in 100 µl of Serum Free Medium.
Mix the solutions gently by carefully pipetting one time.
Combine the two solutions without any mixture procedure (shear stress
may destroy the DNA lipid complex!) and incubate at room temperature
for 15 – 20 min.
After incubation time add as soon as possible the DNA -lipid complexes
dropwise to the cell suspension and swirl the flask with extreme care. If
toxicity is a problem because of very sensitive cells, remove the
transfection mixture after 3 – 6 hours and replace it with medium.
Lipofectamine
One day before transfection, plate 1-5 x 105 cells in 2 ml of full growth
medium so that they will be 90-95% confluent at the time of
transfection.
For each transfection sample, prepare DNA-Lipofectamin 2000
complexes as follows:
Dilute ~ 2 µg DNA in 250 µl of Serum Free Medium (SFM), Mix gently.
Mix Lipofectamin 2000 (twice the amount of DNA) gently before use,
then dilute the appropriate amount in 250 µl of SFM. Mix gently and
incubate for 5 minutes at room temperature. Note: combine the diluted
Lipofectamin 2000 with the diluted DNA within 30 minutes. Longer
incubation times may decrease activity.
After the 5 minute incubation, combine the diluted DNA with the
diluted Lipofectamin
2000 (total volume is 500 µl). Mix gently and incubate for 20 minutes at
room temperature to allow the DNA-Lipofectamin 2000 complexes to
form.
Add the 500 µl of DNA-Lipofectamin 2000 complexes to each well
containing cells and medium. Mix gently by rocking the plate back and
forth.
Incubate the cells at 37°C in a CO2 incubator for 24-48 hours until they
are ready to assay for transgene expression. It is not necessary to
remove the complexes or change the medium; however, growth
medium may be replaced after 4-6 hours without loss of transfection
activity.