Isolation of protein complexes with ZZ fusion proteins


Rationale

Interacting proteins are isolated from lysates with a bait protein that is coupled to a IgG-Sepharose matrix via a IgG-ZZ complex. In   contrast to immunprecipitation or endogenous expression of tagged proteins this approach also works with less abundant bait proteins.


isolation of recombinant ZZ-fusion protein

  • expression in BL21DE
  • purification by Ni chelate chromatograpy
  • dialysis against binding buffer
  • add glycerol to 50% or sucrose to 250 mM
  • freeze aliquots of 50-100 µg

prepare a cleared lysate

  • lyse embryos in extraction buffer
  • extract lipids with trichlor-trifluor-ethan
  • clear the lysate, 40k, 30 min UZ
  • determine the protein concentration (Bradford or A280 with A(1 mg/ml) = 1,0)

binding of the ZZ-fusion protein to IgG beads

  • wash 10 µl of IgG sepharose beads with binding buffer
  • add 10-50 µg of ZZ fusion protein to the beads, incubate on the wheel for 1h, 4°C
  • wash 3x with binding puffer, spin beads 2 min, 1k

binding and analysis

  • add 500 µl of lysate to the beads in extraction buffer
  • incubate on the wheel overnight
  • wash 3x with washing buffer
  • transfer into small spin columns with new filter
  • wash 1x with washing buffer
  • add 40 µl of elution buffer, incubate 15 min at RT
  • spin 2 min, 2k, collect the eluate
  • repeat elution
  • pool the eluates (80 µl)
  • add 1,5 ml 2-propanol to precipitate the proteins
  • incubate for 1 h on ice
  • spin 15 min, 4°C, 13k
  • discard the supernatant
  • wash the pellet with 100 µl ethanol
  • dry in speed-vac
  • dissolve the pellet in 20-40 µl Lämmli, also add Lämmli to the beads to check for uneluted proteins
  • analyse 10 µl of the fractions by SDS-PAGE
  • silverstaining or collidal coomassie for MS analysis

parameters:

lysate:  the proteins can be extracted with low salt (50-100 mM NaCl) or high salt (500 mM NaCl). Detergents may be added for better extraction (0,l% Tween, 0,1% Triton, desoxycholate, ...)
 ZZ-fusion protein: Using a purified ZZ fusion protein allows quantification of the amount bound to the ZZ beads. Alternatively the IgG beads may be added to the bacterial lysate. Background bands may disturb further analysis, however.

binding buffer: 

50 mM Tris/HCl pH 7
500 mM NaCl

washing buffer:

50 mM Tris/HCl pH 7
100 mM NaCl  (depending on conditions)

elution buffer:

50 mM Tris/HCl pH 7
1,5 M MgCl2
100 mM NaCl