Filter Binding Assay for EBNA-1

Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School http://mcardle.oncology.wisc.edu/sugden/Protocols/html files/Filter Binding Assay.htm Last Modified 5.2.2003 by Scott E. Lindner
Wash one 0.45um Millipore nitrocellulose filter (Cat# HAWP01300) in 1ml 0.4M KOH for 1 min; wash in 3ml dH₂O for >1min.

Assemble filter apparatus: white plastic bottom support/funnel, nitrocellulose filter, white plastic top with syringe adapter, 3ml syringe

Pre-incubate filter with 100μl Binding Buffer at filter binding temperature (4℃) until ready to bind DNA /EBNA-1 solution (e.g. conduct in 7th floor cold room).

Incubate DNA fragment of interest with DNA with no known binding sites in a total volume of 100-250μl Binding Buffer.

Incubate 4μg 6xHis-dnEBNA-1 in total volume of 100μl Binding Buffer.

Pre-incubate both the DNA and Protein solutions at 37℃ for 5min.

Mix the DNA and Protein solutions by pipetting, incubate for 15min, 37℃. Shift solution to binding temperature by addition of ice cold Binding Buffer to a final volume of 1000ul, incubation on ice until loading on filter

Pass pre-incubation through filter by increased pressure supplied by syringe above filter. Remove syringe from filter apparatus.

Load DNA /EBNA-1 solution in upper portion of the filter apparatus, reattach syringe above. Filter all of solution through at slow rate (1ml/1min). Remove syringe and withdraw plunger from syringe. Reattach syringe. Reload flowthrough into filter apparatus and incubate at filter binding temperature (4℃) for 10min. Pass through filter as above and collect in a labeled tube.

Load 400μl Wash Buffer into the syringe. Reattach the plunger and wash the filter immediately, collecting fraction in a separate tube. Remove syringe and withdraw plunger from syringe. Reattach syringe.

Repeat wash as per step 10, but pass through the filter when you are immediately ready to add elution buffer

Shift membrane to elution temperature (RT). Remove syringe, load 100μl elution buffer to top chamber of filter apparatus. Reattach syringe and allow the solution to incubate for 10min at elution temperature (RT). Pass through filter, repeat with a second 100μl elution.

Add 1/10 volume 3M Sodium Acetate, 1μl glycogen (20ug/μl stock), and 2-3 volumes 100% EtOH, mix by inversion

Spin 30min, 4℃. Decant supernatant, invert on sterile Kimwipe.

Spin 5min, 4℃. Pipette residual supernatant, allow to partially dry. Resuspend in 20μl 1xTE.

Load on 0.8% TAE agarose gel, visualize by EtBr staining by UV.

Quantify percent of each band present by comparison to input DNA fragment used.

(Optional) Quantify protein in each fraction by SDS-PAGE/Western.

Binding Buffer

10mM HEPES

10mM MgCl₂

150mM NaCl

Add 5% DMSO fresh

Wash Buffer:

10mM HEPES

10mM MgCl₂

150mM NaCl

10% glycerol

Add 5% DMSO fresh

Elution Buffer:

10mM HEPES

150mM NaCl

0.2% SDS