Michael Rehli • Dept. Internal Medicine III • University Hospital • F.-J.-Strauss Allee 11 • 93053 Regensburg
fon: **49 (0)941 944 5587    fax: **49 (0)941 944 5593

Methods


    Available Protocols
  1. DEAE-Transfection of THP-1 cells
  2. Production of MBD-Fc protein
  3. Methyl-CpG Immunoprecipitation (MCIp)



DEAE-Transfection of THP-1 cells (transient) [pdf]

Reagents:

STBS    
3.029 g (25 mM) Tris/HCl, pH 7.5
8.01 g (137 mM) NaCl
0.373 g (5 mM) KCl
0.161 g (0.6 mM) Na2HPO4·7 H2O
0.103 g (0.7 mM) CaCl2·2H2O
0.102 g (0.5 mM) MgCl2·6H2O
  add H2O to 1 l, autoclave
DEAE-Dextran    
10 mg/ml in STBS  
  filter sterilize (0.2 µm) and store at RT
PBS    


Protocol:
The day before transfection, a sufficient number of THP-1 cells to allow duplicate transfection for each reporter plasmid is seeded at 350000 cells/ml into tissue culture flasks. On the following day, for each transfection 6 ml cell suspension are centrifuged (8 min. 300xg, 4°C) the cell pellet washed twice with 5 ml STBS each, and the supernatant is removed completely. For each transfection, 200 ng of the luciferase reporter plasmid and 20 ng of the renilla control plasmid in 70 µl STBS are combined with 70 µl DEAE-Dextran (800 ug/ml), mixed and immediately added drop-wise directly onto the THP-1 cell pellets. The cells are incubated at 37°C for 20 min, washed twice with 5 ml STBS each, resuspended in 6 ml 10% FCS/RPMI and cultured in 60 mm tissue culture dishes. Cells are harvested after 48h, the culture dishes washed once with 5 ml PBS at RT and the cells are pelleted (10 min , 400xg, 4°C). Pellets are washed once with 10 ml PBS and the PBS removed completely by decanting and briefly inverting the centrifuge tubes onto Kleenex paper towels. Cells are lysed and assayed.
Note: This protocol is only suited for transient transfections. For stable transfections we have sucessfully used the commercially available kit from AMAXA.







Production of MBD-Fc protein [pdf]

Reagents:

Insect-Xpress medium (Bio Whittaker)
Hygromycin (Invitrogen)
1M CuSO4 in H2O (sterile)
Bindung buffer (10xTBS)
151.4 g (500 mM) Tris/HCl pH 7.4
219.2 g (1.5 M) NaCl
9.3 g (10 mM) EDTA
125 mg (0.05%) NaN3
  add H2Obidest to 2.5 l
Elution buffer
2.9 g (0.1 M) Citric acid (pH 3.0)
  add H2Obidest to 100 ml
Neutralization buffer
18 g (1.5 M) Tris/HCl (pH 8.8)
  add H2Obidest to 100 ml


Protocol:

Expansion of the MBD-Fc expressing Drosophila S2 cells in tissue culture flasks
MBD-Fc S2 cells are maintained at a density of 1-4 million cells/ml in Insect-Xpress medium (Bio Whittaker) plus 400 µg/ml hygromycin (Invitrogen), (no FCS) in an incubator at 21°C. The cells should be splitted all 3-5 days (tapping, seeding in new cell culture bottles, adding new medium). Notice: Cell density should not increase above 15 million cells/ml!

Transferring cells into roller bottles
For large scale protein production 3-4 million cells/ml are transferred in 100-400 ml Insect-Xpress plus 400 µg/ml hygromycin into 2000 ml roller bottles and cultured for 3 5 days. The cell density should not increase above 10 million cells/ml! In case, add medium. Leave about 1/10 of the culture in tissue culture flasks (expansion culture) to be able to change the producing cells periodically.

MBD-Fc protein production
For protein production, the medium is changed. The suspension cells are spinned down (without detaching the adherent fraction) and transferred with a density of 10-15 million cells/ml in Insect-Xpress w/o hygromycin but with 0.5 mM CuSO4 back into the same roller bottles. The MBD-Fc peptide-containing culture medium is harvested after 4 days of culture (production phase). Subsequently, cells are recultured in Insect-Xpress with a density of 3-4 million cells/ml plus 400 µg/ml hygromycin w/o CuSO4 for 3 days in the same bottle (selection phase) (maximum of 400 ml/roller bottle). After each selection phase, we start another production phase (4 days) for continued protein production (always in the same bottle. Make sure cells don’t get too dense (>20 million cells/ml)). This is continued until production of S2 cells gets inefficient (1 liter production phase medium should contain up to several mg (2-6 mg) of the protein).

Protein purification
Cell culture supernatants of production phases are combined and remaining cells and debris are removed by centrifugation at 2000xg, 20min, 4°C and subsequently at 15-20000xg, 40 60 min, 4°C. (Supernatants can be stored for several days at 4°C). Supernatants are dialyzed against TBS (pH 7,4) (at least 48h at 4°C; replace TBS 3-4 times). The MBD-Fc protein is affinity-purified using protein A sepharose. Elution is performed in 1 ml fractions with elution buffer into 1.5 ml tubes each containing 50 µl neutralization buffer. The protein containing fractions are combined and dialyzed against TBS or PBS. We add 0.2% gelatin and 0.02% sodium azid for long term storage at 4°C.







Methyl-CpG Immunoprecipitation (MCIp) [pdf]

Reagents:

MBD-Fc protein
Protein A Sepharose 4 Fast Flow beads (Amersham) or equivalent
2 ml Ultrafree®-MC centrifugal filter devices (Amicon/Millipore)
Adhesive Film
Buffers: (for three large-scale MCIp):
TME (10x)
4 ml (200 mM) Tris/HCl (1M) pH 8.0
400 µl (20 mM) MgCl2 (1M)
200 µl (5 mM) EDTA (0.5 M) pH 8.0
  add H2Obidest to 20 ml
Buffer A (300 mM NaCl)
1 ml (1x) TME(10x)
600 µl (300 mM) NaCl (5M)
100 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 10 ml (8.3 ml)
Buffer B (400 mM NaCl)
400 µl (1x) TME(10x)
320 µl (400 mM) NaCl (5M)
40 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 4 ml (3160 µl)
Buffer C (500 mM NaCl)
400 µl (1x) TME(10x)
400 µl (500 mM) NaCl (5M)
40 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 4 ml (3240 µl)
Buffer D (600 mM NaCl)
400 µl (1x) TME(10x)
480 µl (600 mM) NaCl (5M)
40 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 4 ml (3080 µl)
Buffer E (1000 mM NaCl)
400 µl (1x) TME(10x)
800 µl (1000 mM) NaCl (5M)
40 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 4 ml (2760 µl)
Buffer X (300 mM NaCl endconc.; for 400 µl sonicated DNA in TE or water)
600 µl (1x) TME(10x)
360 µl (300 mM) NaCl (5M)
60 µl (0.1 %) NP-40 (10 %)
  add H2Obidest to 4.8 ml (3780 µl)


Protocol:

Day 1: Incubation of MBD-Fc protein with Sepharose beads
Add 200 µl sepharose beads/sample into a 15 ml Falcon tube containing 10 ml of TBS buffer and mix by gently inverting several times. Add purified MBD-Fc protein (80-100 µg/sample). Fill the tube with TBS buffer to the top. Seal and incubate over night on a rotator at 4°C.
Optional: Sonification of genomic DNA:
Sonicate your genomic DNA sample to the desired fragment size (e.g. 5 µg in 500 µl TE). For comparing two different samples, make sure that the degree of fragmentation is equal! Check by agarose gel electrophoresis! Measure the DNA concentration.
If the genomic DNA is fragmented using restriction enzymes, make sure that the digest is complete.

Day 2: Precipitation
Notice: Binding of mCpG-DNA depends on the fragmentation size and MBD-Fc coating density on the sepharose beads. We strongly recommend performing a test fractionation with every large column experiment, to make sure that the salt concentration selected for recovering methylated DNA really contains the desired mCpG density. We do the test MCIp in the morning, run a real-time PCR for control genes like the imprinted SNRPN gene and decide on which salt concentration is then used for all samples that are fractionated in the afternoon.
Also notice that the buffer compositions are just a guide line. The salt concentrations may need adjustment to your own protocol and conditions!


MCIp Procedure:
  • Disperse the MBD-Fc coated beads equally into 2 ml Ultrafree®-MC centrifugal filter devices.
  • Spin wash twice with 2 ml buffer A. Discard flow through.
  • Seal the bottom hole of the spin tubes using adhesive tape; (This way, beads don’t have to be transferred after incubation)
  • Re-suspend beads in 1600 µl buffer X;
  • Add sonicated gDNA (4 µg in 400 µl TE) and rotate for 2-3 hours at 4°C;
  • Remove the sealing and transfer spin tubes into fresh 15 ml tubes;
  • Centrifuge at 5000 RPM for 1 min at 4°C. Keep the flow-through if required. Transfer spin tubes into fresh 15 ml tubes.
  • Wash spin tubes twice with 600 µl buffer B (5000 RPM, 1 min, 4°C; invert spin tubes twice after buffer was added). Keep the flow-through if required. Transfer spin tubes into fresh 15 ml tubes.
  • Wash spin tubes twice with 600 µl buffer C (5000 RPM, 1 min, 4°C; invert spin tubes twice after buffer was added). Keep the flow-through if required. Transfer spin tubes into fresh 15 ml tubes.
  • Wash spin tubes twice with 600 µl buffer D (5000 RPM, 1 min, 4°C; invert spin tubes twice after buffer was added). Keep the flow-through if required. Transfer spin tubes into fresh 15 ml tubes.
  • Elute twice with 600 µl buffer E (5000 RPM, 1 min, 4°C; invert spin tubes twice after buffer was added). The combined flow-through contains all remaining bound material. For further processing, the 1200 µl eluate needs to be de-salted. We routinely use the QIAquick PCR Purification Kit and elute twice with 50 µl elution buffer (@55°C).

Control Run SNRNP
Figure:
Example of a control run with human SNRPN primers (see Gebhard et al. 2006, Cancer Res.). The unmethylated allele elutes mainly at 300 mM, the methylated from 550 mM.


MCIp procedure (for conventional columns, 10 – 500 ng sonicated DNA)
The procedure is basically scaled down from the large column protocol. We use 40 µl beads per MCIp (15-20 µg MBD-Fc protein), and run the procedure in the small Millipore columns. The initial buffer volume is 350 µl and beads are washed twice with 175 µl of individual buffers. All fractions are desalted and quantified (in comparison with the starting material) via real-time PCR.