Protein-DNA Binding Assays

EMSA

Preparing of cell extracts

Annealing of oligos

Labeling of annealed oligo with ³²P-alpha-dATP with TdT

Incubation of extracts with oligos and native PAGE
 

ABCD assay

EMSA:

Preparing of cell extracts

Cells (e.g. 293 cells) of one 6-well (10 cm², app. 10⁶ cells): add 100 µl/well:

1x EMSA lysis buffer (stock sol.: 5x):

• 10 mM Tris/HCl pH 7.5

• 1 mM EDTA

• 5 mM MgCl

• 50 mM KCl

• 1 mM DTT

• 1x protease inhib. (Complete)
  
  

Ø  Lysis of cells by 4 freeze/thaw cycles (-80°C/37°C incubator): on the plates,

Ø  check breakage by microscopy

Ø  suspend with pipette and transfer to Eppendorf tubes > 1 additional freeze/thaw cycle

Ø  centrifugation: 14 000 rpm, 4°C, 15 min > take supernatant: measure vol.: approx.75 µl
( > can be frozen at -70°C)

Ø  add glycerol to 10% final conc., add KCl to 150 mM (4.2 µl 1 M to 75 µl sample)

Determine protein concentration of the extracts with Bradford reagent:

standards: BSA: 0, 1, 2, 3, 4 µg (µl) in 96well plates

extracts: 1 µl each (or diluted); 

+ Biorad Bradford reagens (1:5, 200 µl) > measure OD595 in a microtiter plate reader

expected concentration of extracts: 2 – 3 µg/µl

Annealing of oligos

Ø  equimolar amount of sense and antisense oligo: 400 pmol each (approx. 5 µg): 4 µl

Ø  20 µl 10x Buffer B (Roche)

Ø  A.dest. ad 200 µl (172 µl)

Ø  heat to 95°C (5 min)

Ø  switch off the thermoblock and let cool down to RT

concentration: 400 pmol/200 µl = 2 pmol/µl

Labeling of annealed oligo with ³²P-alpha-dATP with TdT

(Fermentas #EP0161, Terminal deoxynucleotide Transferase)

• 10 µl 5x reaction buffer

• 2.5 µl annealed oligo (10 pmol of 3’ termini = 5 pmol ds-oligo)

• 5 µl ³²P-alpha-dATP (10 µCi/µl > 50 µCi)

• 2 µl TdT (40 u)

• 30.5 µl A.dest. nuclease free

incubate at 37°C for 15 min
(Stop the reaction by heating to 70°C for 10 min).

Incubation of extracts with oligos and native PAGE

(Electrophoresis on PhastSystem, GE Healthcare, formerly Pharmacia-Amersham: see instruction manual of the manufacturer)

-       2 µl extract,

-       1 µl 1x EMSA lysis buffer (or competitor > 20x molar excess)

-       0.5µl ³²P-Oligo

> incubated 15 min at RT

+ 1 µl 1x EMSA lysis buffer (+ small amount bromphenolblue)

-       pipetted into 4 µl sample combs of the PhastSystem

-       run samples on 12.5% homogenous Phastgels using native buffer strips

Sample Appl. down at 4.2.        0Vh

Sample Appl. up at 4.2                         2Vh

Step 4.1. 400 V            10.0 mA           2.5W    15°C     10 Vh

Step 4.2. 400 V              1.0 mA           2.5W    15°C       2 Vh

Step 4.3. 400 V            10.0 mA           2.5W    15°C     140 Vh

Example:

ABCD-Assay (Avidin-Biotin Complex with DNA)

(1)   Preparations

Buffer H

HEPES pH 7,8-7,9:    - 1 M solution use  NaOH to adjust pH 7,8-7,9

-   sterile filter all solution used

-   The stringency of the assay is dependent on KCL concentration: High stringency: 150mM or 250mM KCL

-   For endogenous protein 50mM KCL appears to work best

(2)   Streptavidin-Agarose

-          Re-suspend streptavidin-agarose by vortexing, take out 500 µl (mark with a pen), centrifuge and discard supernatant

-          Wash three times with 1000 µl Buffer H (50mM KCL, +5mg/ml BSA), washing: re-suspend, 10min on rotating well (4°C), centrifuge (4°C), discard supernatant

-          Add 1 ml Buffer H (+5mg/ml BSA, +10 µl 10mg/ml SS DNA) incubate 24 h on rotating well (4°C)

-          wash three times with 1000 µl Buffer H (50mM KCL, without BSA)

-          add up to (50mM KCL, without BSA) 500 µl (use mark)

(3)   Anneal oligonucleotides

50 µg S-Oligo

50 µg AS-Oligo

10 µl 10x Annealing Buffer

Add to 100 µl with H₂O

10x Annealing Buffer:   0,5 M NaCl

                                      0,2 M Tris pH 7,4

-          Incubate on heating block for 5min at 95°C

-          Switch of heating block, let sit until block has cooled to 30°C to 40°C

-          Store oligos at 4 °C or -20 °C

Example sequence of NFkB oligo (for Pulldown)

(red: one NFKB consensus sequence)

Competitor oligo: same oligo without biotin

(4)   Preparation of cell lysate

NETN  

Sterile filter NETN solution.

Scrape cells in PBS with cell scraper.

Centrifuge cells: 5´ 1500 rpm

Re-suspend pellet in 1ml NETN (for 75 cm² flask)

Sonify:

6 pulses á 2 s, 30% power

centrifuge: 14 000 rpm, 4°C, 15 min

Keep supernatant.

(5)   Assay

Pulldown

200 µl  Cell Lysate

2 µl biotyniliated oligo (1 µg/µl)

2 µl SS-DNA (10 mg/ml)

200 µl Buffer H

Competitor control

200  µl  Cell Lysate

2 µl biotyniliated Oligo (1 µg/µl)

2 µl SS-DNA (10 mg/ml)

200 µl Buffer H

20 µl not biotyniliated oligo (1 µg/µl)

Negative control

200 µl  Cell Lysate

2 µl SS-DNA (10 mg/ml)

200 µl Buffer H

Positive/Input control (for WB)

20 µl Cell Lysate

(if cell lysate is less than 200 µl add to 200 µl with Buffer H, successful PD will depend on initial protein concentration vs stringency)

à incubate 5 min at 37 °C in heating block shaking (1100 rpm)

à incubate for 1 h on ice

-          add 40 µl of  prepared streptavidin-agarose

à incubate 30 min at 4 °C on rotating wheel

-          centrifuge at 4°C (13.200rpm, 2 min)

-          wash 5 times with 1 ml Buffer H (50 mM, 150mM or 250mM KCl)

washing: remove supernatant, add 1 ml Buffer H, incubate 10 min at 4 °C on rotating wheel, centrifuge at 4°C (13.200rpm, 2 min)

-          after last wash remove all remaining Buffer H: you can use the tips for blot loading to avoid sucking in the beads

-          add 25 µl 1x Western Blot loading Buffer

optional: you can freeze your samples at this point

-          Blot loading

à incubate 3 min at 95 °C shaking (1100 rpm)

-          Centrifuge (13.200rpm, 2 min), use 20 µl for loading

-          for Input control use 20 µl cell lysate + 4 µl loading buffer

Products:

Strepavidin-Agarose (Heidelberg)              - Novagen Cat.# 69203-3

Strepavidin-Sepharose (ab HU + MS-275) - GE Healthcare # 17-5113-01