azim58 - dot blot protcol


Dot Blot Protocol

Abcam dot blot protocol
http://www.abcam.com/ps/pdf/protocols/Dot%20blot%20protocol.pdf

Description
A technique for detecting, analyzing, and identifying proteins, similar
to the western blot technique but
differing in that protein samples are not separated electrophoretically
but are spotted through circular
templates directly onto the membrane or paper substrate.
Concentration of proteins in crude preparations (such as culture
supernatant) can be estimated semiquantitatively
by using “Dot Blot� method if you have both purified protein and
specific antibody against it.

Reagents
TBS:
20 mM Tris-HCl
150 mM NaCl
pH 7.5
TBS-T:
0.05% Tween20 in TBS
BSA/TBS-T:
0.1% BSA in TBS-T
Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.)


Procedure

* Have nitrocellulose membrane ready, draw grid by pencil to indicate
  the region you are going to blot (see
below).

* Using narrow-mouth pipet tip, spot 2 µl of samples onto the
  nitrocellulose membrane at the center of the
grid. Minimize the area that the solution penetrates (usually 3-4 mm
diam.) by applying it slowly.

* Let the membrane dry.
* Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr,
  RT). Use 10cm Petri Dish for reaction
chamber.


o It is possible that even better blocking can be achieved if the
  membrane is blocked overnight at 4 C.
* Incubate with primary antibody (0.1-10 µg/ml for purified antibody,
  1:1000 to 1:100000 dilution for
antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in 5%
BSA/TBS-T for 1 hr at RT.

* Wash three times with TBS-T (3 x 5 min).
* Incubate with secondary antibody 1:10,000 in 5% BSA in TBST (for
  optimum dilution, follow the manufacturer’s
recommendation) for 1 hr at RT.

* Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with
  TBS (5 min).
* Dry membrane (can be dried between blotting filter paper)
* If the secondary antibody was an HRP antibody, then incubate with an
  HRP reagent.
  o If an ECL reagent was used then incubate for 1 min, cover with
    Saran-wrap (remove excessive solution from the

surface), and expose X-ray film in the dark room. Try several different
lengths of exposure.

* Detect antibody. If an AF647 secondary antibody was used, then the
  antibody can be detected on the Typhoon.
* May want to compare the signal from your unknown sample to that of
  standard and estimate the concentration.