בס"ד

Lysate Preparation:

  1. Prepare the lysis buffer according to the following table:

Lysis Buffer / for 10ml / for 350 ml / for 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

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.

  1. Scratch cells or trypsinize cells for 5 minutes.
  2. If you trypsinize cells, neutralize trypsin with 1:1 ratio medium.
  3. Centrifuge cells 5 minutes at 1.5 rpm.
  4. Aspirate supernatant.
  5. Add DTT to lysis buffer containing PI (1µl 1M DTT : 500µl lysis buffer).
  6. Lyse cells with ice-cold lysis buffer. Pipette until cells thoroughly mixed.
  7. Put cells on ice for 1/2 hour.
  8. Centrifuge cells at max speed (13,000rpm) for 20 minutes at 4 C.
  9. Transfer supernatent to clean eppendorf.
  10. Prepare bradford reagent (1ml Bradford: 4 ml DDW).
  11. Check protein concentration (use triplicates):

by spectrophotometer - 1ml bradford : 1µl protein lysate.

by ELISA – 250 µl bradford: 1 µl protein lysate.

  1. 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:

  1. Prepare the relevant Acrylamide gel according to following table:

Resolving gel:

Stacking gel:

Important:

A.Add the TEMED at the chemical hood!!!

B.Add the APS last. Load the gel immediately after adding the APS.

  1. 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.
  2. Dilute the protein samples with sample buffer(SB)*. The final concentration of the SB need to be X1.
  3. Boil the protein samples for 5 minutes.
  4. 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).
  5. 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.
  6. 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 15.5ml

SDS (danger to breath) 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 transferX10).
  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.

  1. 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.
  1. 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 DNAin 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.