NUCLEAR EXTRACTION OF TISSUE CELLS

This procedure was developed for HNEK cells grown in KGM, from Clonetics.  All steps are performed on ice, with ice-cold reagents, in pre-chilled centrifuge tubes; this is to retard the activity of any proteases present as much as possible.

1. aspirate medium
2. wash with ice-cold PBS
3. add 3 mL ice-cold PBS and scrape cells gently into 15mL tube
4. centrifuge 5 minutes at 500 rpm, 4^OC
5. carefully aspirate supernatant with pipet
6. resuspend pellet in 1 mL ice cold BUFFER I
7. incubate 15 minutes on ice to allow cells to swell
8. add Igepal-CA630 to 1% (100 ul of a 10% stock solution)
9. vortex 10 seconds
10. centrifuge 2-3 minutes at maximum speed (~15K RPM)
11. carefully aspirate supernatant with pipet; this is the cytoplasmic fraction
12. resuspend the pellet in 175 ul ice cold BUFFER II
13. vortex 30 seconds; rotate vigorously at 4^OC 30 minutes
14. centrifuge 15 minutes at maximum speed; remove the supernatant to a fresh, chilled tube. leave on ice if using immediately, or store aliquots at –80^OC until use.  DO NOT freeze/thaw.
15. assay for protein concentration (Pierce’s BCA kit). 

You will need:

• PBS, tissue-culture grade
• 10 % Igepal-CA630 (w/v in dH₂O)
• tissue culture scrapers

A. 100 mL Buffer I – membrane lysis

• 1 mL 1 M Hepes, pH 8.0
• 150 μl 1 M MgCl₂
• 1 mL 1 M KCl
• 100 μl 1 M DTT
• dH₂O to 100 mL

*filter sterilize and store at 4^OC. 
*aliquot enough for one use and add protease inhibitors (1000:1)

B. 100 mL Buffer II – nuclear envelope lysis

• 2 mL 1 M Hepes pH 8.0
• 150 μl 1 M MgCl₂
• 25 mL glycerol
• 42 mL 1 M NaCl
• 40 μl 0.5 M EDTA
• 100 μl 1 M DTT
• dH₂O to 100 mL

*filter sterilize and store at 4^OC. 
*aliquot enough for one use and add protease inhibitors (1000:1)

DESIGN AND LABEL OLIGOS

Choose appropriate oligos corresponding to DNA binding sequence of interest; order and store single strands separately until after labeling (dsDNA usually does not biotinylate with good efficiency using this kit, although there are some ways to increase the efficiency; consult their tech support).

Biotinylate oligos using Pierce DNA biotinylation kit.  Label 5 pmoles ssDNA according to instructions in kit. 

You will need:

• 1 μM solution of oligo to be labeled
• 37^OC temp block
• 0.2 M EDTA
• chloroform:isoamyl alcohol (24:1)

Determine labeling efficiency using the recommended dot-blot setup; process diluted samples and controls through Bio-Rad’s vacuum blot apparatus (slot blotting system).

You will need:

• Neutral nylon membrane
• Blotting apparatus
• TE buffer (10mM Tris.HCl; 1mM EDTA, pH 8.0)
• 96-well microplate; not high-absorption
• UV cross-linker

DNA detection solutions and detection method ( NEB Phototope-Star kit):

A.      1 L blocking solution / 10X wash solution I

• 7.3g NaCl,
• 2.4g Na₂HPO₄
• 1g NaH₂PO₄
• 50g SDS
• dH₂O to 1 L

B.   1 L wash solution II (10X)

• 12g tris base
• 5.8g NaCl
• 2g MgCl₂
• pH to 9.5 with HCl
• dH₂O to 1 L

Using Pierce’s guidelines, dilute samples and controls in a 96-well dish; rinse membrane in TE buffer. Assemble apparatus and wash wells with 200 ul TE.  Load samples into apparatus.

After blotting diluted oligo samples onto the membrane, remove membrane from apparatus and allow to dry briefly (~2-3’) on a paper towel or piece of filter paper.  UV Cross-link the DNA onto the membrane.  I use the ‘autocrosslink’ function on the crosslinker (Stratalinker; countdown from 120mJ/cm²). Proceed with the membrane through the DNA detection steps (based on kit instructions with NEB Phototope-Star):

1. rinse in blocking solution 5’ RT
2. incubate with streptavidin (1mg/mL stock, 1:1000, 5μl/5mL blocker) 5’ RT
3. rinse with Wash I  X 2, 5’ RT
4. incubate with biotin-alkaline phosphatase-conjugate (1:1000, 5μl/5mL blocker) 5’ RT
5. rinse with blocker 5’ RT
6. rinse with Wash II X 2, 5’ RT
7. incubate with detection reagent  5’ RT
1. prepare 1X CDP assay buffer (1:25 in H₂O)
2. add CDP-Star reagent to assay buffer at around 1:400 (for example, 15 μl/6mL buffer)
8. drain reagent, wick excess moisture by briefly blotting on a paper towel, detect with X-ray film

Add approximately equal amounts of + and – complimentary oligos to obtain labeled dsDNA probes.  To anneal, heat to 90^OC and incubate for ~10 minutes.  Cool slowly to RT.  Use immediately or store at 4^OC for the short term, -80^OC for the long term.

BINDING REACTION & ELECTROPHORESIS

Use approximately 0.1 to 0.3 pmoles each oligo strand (1-3μl per oligo label reaction). This is assuming labeling efficiency ~70% to 80%, as calculated with the dot blot.  For competition controls, use 100-fold excess of unlabeled oligo mixture in addition to other binding reaction components.

Prepare a 1%-1.4% agarose gel in TAE (1X) or TBE (0.5X) with the appropriate number of lanes.  It is not necessary to add ethidium bromide or to photograph the gel after running.

You will need:

• 1 % Igepal-CA630 (w/v in dH₂O)
• 1 mg/mL poly (dI.dC) in 10mM tris, 1mM EDTA, pH 7.5

A. 20 mL blue juice stock, ~6X

• 0.05 g bromophenol blue
• 8 g sucrose
• dH₂O to 20 mL
• *fs and store at 4^OC

B. TAE, 50X

• 84.7 g tris base
• 20 mL glacial acetic acid
• 35 mL 0.5 M EDTA
• dH₂O to 350 mL
• *use at 1 X

C. binding buffer, 5X

• 1 mL 1 M Hepes pH 8.0
• 2.5 mL 1 M KCl
• 25 μl 1 M DTT
• 5 μl 0.5 M EDTA
• 50 μl 1 M MgCl₂
• 2.5 mL glycerol
• dH₂O to 10 mL

*aliquot into small volumes and store at –20^OC.

Set up binding reactions as follows:

• 4 μl 5X binding buffer
• 2 μl 10X poly dI.dC
• 1 μl 1% Igepal-CA630
• X μl oligo pair
• X μl nuclear extract (to add 1 μg to each reaction)
• X μl H2O to 20 μl total volume

Assemble components on ice.  Incubate at RT 15-20 minutes.  Add ~3-5 μl blue juice to each tube and add the entire sample to one gel lane.  Run the gel ~40V over several hours (lower voltage equals slightly better resolution); the blue juice corresponds approximately to the location of the oligo controls, so do not allow the blue juice to run off the gel.  Proceed immediately to the transfer step.

*note: for supershift assay, use 1ul antibody of interest per reaction, adjusting the volume of water accordingly.  Assemble the components on ice without adding oligo; incubate on ice 10’, then at RT 20’, then add oligo and incubate an additional 10’ at RT.  This allows sufficient time for the antibody to bind the transcription factor / subunit (determined empirically with p65 and p50 subunits of NF-κB, 2005).  I would like to note that some antibody-transcription factor complexes will not bind to the appropriate DNA sequence due to conformational alterations in the bound form; with some antibody-transcription factor-DNA interactions it is necessary to add the oligo to the nuclear extract, then pre-incubate, then add the antibody.  In my experience, using my methods, this did not work and so I add the antibody first and preincubate with the nuclear extract before adding the oligo.

DNA TRANSFER AND BLOTTING

After electrophoresis, transfer DNA a la Maniatis’ Southern methodology (9.34, v 2, Molecular Cloning Manual). 

You will need:

A.      denature

• 87.66 g NaCl
• 20 g NaOH
• 800 mL dH₂O

*adjust volume to 1 L and autoclave

B.      neutralizer

• 121.1g tris base
• 700 mL dH₂O
• *adjust pH to 7.4 with HCl (~70mL)
• 87.66 g NaCl

*adjust volume to 1 L and autoclave

C.      20 X SSC (use at 10X)

• 175.3 g NaCl
• 88.2 g Sodium Citrate
• 800 mL dH₂O

*adjust pH to 7 with NaOH
*adjust volume to 1 L and autoclave

Incubate gel in denature buffer at 25^OC for 30’, on a platform shaker.   Rinse briefly with dH₂O.  Equilibrate with neutralizer at 25^OC for 30’, then change neutralizer and incubate at 25^OC for an additional 15’.  Equilibrate membrane in 10X SSC briefly (~5’).  Assemble capillary transfer apparatus as indicated by Maniatis.  Allow transfer to proceed through a short overnight. 

The following day, UV crosslink the nylon membrane and proceed to detection, exactly as with the dot blot performed above.

Please note that this is a method that works for me, and many different factors and variables may need to be adjusted for your system.  I have found that Pierce’s website is a fantastic technical resource for non-radioactive EMSA.

Please also note that in some places many details are skipped, assuming prior knowledge/experience, or that the appropriate kit instruction manuals (noted in the text) are the best places to get information.