CROSSLINKING OF PROTEINS

1
CROSSLINKING OF PROTEINS

• REFERENCES  Reithmeier, R.A.F. and Bragg, P.D.
  (1977). Biochim Biophys Acta. 466245-256. Angus
  and Hancock. (1983). Bacteriol. 1531042-51.  

Kris-itd.unair.ac.id (for education purpose only)
2

• MATERIALS
• Outer membranes (or purified proteins) at a
  protein concentration of approx. 2 mg/ml (500
  µg/ml).
• dithio-bis-succinimidylpropionate (DSP) stock
  solution make up in DMSO at 15X the desired
  final concentraion (Final concentration will be
  approx. 2 mg/ml for outer membranes and 0.5 mg/ml
  for purified proteins)
• 0.2 M Triethanolamine buffer pH 8.5
• 1 M Tris-HCl pH 8.5
• gel sample buffer without ß-mercaptoethanol
  (2-ME) (ie. 4 SDS/0.5 M Tris pH 6.8/20
  gylcerol).
• heating block at 100 oC
• 11 ployacrylamide gel
• Second dimension soaking buffer 0.125 M
  Tris-HCl pH 6.8/10 2-ME or 0.125 M Tris-HCl pH
  6.8/10 dithiothreitol.
• 11 gel stacking gel with no wells ( poured to
  3/16 from top of plate). We also have a special
  comb which incorporates two small wells to run
  molecular weight standards or a reference sample
  concurrently.
• 5 ml 0.8 agarose/gel
• NOTE - the number of gels you require for the
  second dimension should be made at the same time
  as the one for the first dimension.

3

• METHOD
• First dimension
• outer membrane and purified protein preparations
  are prepared in 0.015 ml volumes of 0.2 M
  triethanolamine buffer pH 8.5, following the
  specifications of Reithmeier and Bragg (1977).
• Add 1 µl of the crosslinker, DSP, ( dissolved in
  dimethylsulphoxide) to make a 1/15 dilution to
  give the optimun final concentration. For the
  first experiment, a range of concentrations
  should be tried.
• React at room temperature for between 15 s and 2
  min ( also determined by trial). A shorter
  reaction time would be preferable, to minimize
  fortuitous crosslinking of protein.
• Add 5 µl 1 M Tris-HCL pH 8.5 (an excess) to stop
  the crosslinking reaction.
• Dilute 11 into sample buffer containing SDS,
  Tris and glycerol without reducing agent.
• Heat samples at 100oC for ten minutes.
• Electrophoresis in the first dimension as for a
  normal protein gel. It is best to run parallel
  samples for second dimension electrophoresis on
  one side of the gel and those for direct staining
  to visualize the first dimension on the other
  side.

4

• Second Dimension
• Cut strips representing each sample well to be
  re-electrophored from the first dimension gel
  with a razor blade. We have special blades which
  are 5 in. long for this purpose. Make a very
  small notch to mark the bottom end of each cut
  out strip before soaking them.
• Soak the first dimension gel strips in 10
  2-mercaptoethanol (v/v) or 10mM dithioerythritol
  (w/v) in a plastic petri dish for 30 min at room
  temperature, with intermittent agitation.
• Lay the soaked strips carefully atop the second
  dimension gel, being sure to pre-moisten the
  glass of the gel plates with electrophoresis
  buffer so that the strips will slide between the
  plates. Using gloves and a thick spacer, nudge
  the strips into the gap between the plates so
  that the strip is in contact with the second gel
  at all points along its length. Mark which end is
  the top of the strip and which is the bottom on
  the plates.
• Seal the strips atop the second dimension
  SDS-polyacrylamide gel with 0.8 (w/v) agarose
  and electrophorese as normal. A small amount of
  the same sample (or tracking dye) may be added if
  a well was incorporated into the stacking gel.
  This will serve as a reference point to find the
  protein(s) you are interested in (and/or to
  follow the progress of the run).