NUCLEAR EXTRACTION OF TISSUE CELLSThis 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.aspirate medium wash with ice-cold PBS add 3 mL ice-cold PBS and scrape cells gently into 15mL tube centrifuge 5 minutes at 500 rpm, 4OC carefully aspirate supernatant with pipet resuspend pellet in 1 mL ice cold BUFFER I incubate 15 minutes on ice to allow cells to swell add Igepal-CA630 to 1% (100 ul of a 10% stock solution) vortex 10 seconds centrifuge 2-3 minutes at maximum speed (~15K RPM) carefully aspirate supernatant with pipet; this is the cytoplasmic fraction resuspend the pellet in 175 ul ice cold BUFFER II vortex 30 seconds; rotate vigorously at 4OC 30 minutes centrifuge 15 minutes at maximum speed; remove the supernatant to a fresh, chilled tube. leave on ice if using immediately, or store aliquots at –80OC until use. DO NOT freeze/thaw. 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 lysis1 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 4OC.*aliquot enough for one use and add protease inhibitors (1000:1)B. 100 mL Buffer II – nuclear envelope lysis2 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 4OC.*aliquot enough for one use and add protease inhibitors (1000:1)DESIGN AND LABEL OLIGOSChoose 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 37OC 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 I7.3g 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):rinse in blocking solution 5’ RT incubate with streptavidin (1mg/mL stock, 1:1000, 5μl/5mL blocker) 5’ RT rinse with Wash I X 2, 5’ RT incubate with biotin-alkaline phosphatase-conjugate (1:1000, 5μl/5mL blocker) 5’ RT rinse with blocker 5’ RT rinse with Wash II X 2, 5’ RT incubate with detection reagent 5’ RT prepare 1X CDP assay buffer (1:25 in H2O) add CDP-Star reagent to assay buffer at around 1:400 (for example, 15 μl/6mL buffer) 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 90OC and incubate for ~10 minutes. Cool slowly to RT. Use immediately or store at 4OC for the short term, -80OC for the long term.BINDING REACTION ELECTROPHORESISUse 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, ~6X0.05 g bromophenol blue 8 g sucrose dH2O to 20 mL *fs and store at 4OC B. TAE, 50X84.7 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, 5X1 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 –20OC.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 BLOTTINGAfter electrophoresis, transfer DNA a la Maniatis’ Southern methodology (9.34, v 2, Molecular Cloning Manual).You will need:A. denature87.66 g NaCl 20 g NaOH 800 mL dH2O *adjust volume to 1 L and autoclaveB. neutralizer121.1g tris base 700 mL dH2O *adjust pH to 7.4 with HCl (~70mL) 87.66 g NaCl *adjust volume to 1 L and autoclaveC. 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 autoclaveIncubate gel in denature buffer at 25OC for 30’, on a platform shaker. Rinse briefly with dH2O. Equilibrate with neutralizer at 25OC for 30’, then change neutralizer and incubate at 25OC 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.Notes: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.联系人：蚂蚁淘编辑部Email：service@bioon.com电话：021-54485309传真：021-54485087