Biomedical Engineering Reference
In-Depth Information
The activated dry membrane can be stored in a refrigerator for
several weeks.
Blot the proteins onto the affinity membrane either by dot blot
(Protocol 4.8), by electrotransfer (Protocol 2.5.3), or by capillary
blotting (handling analogously to Southern blotting Protocol 2.5.9).
Capillary blotting is recommended, if the gel is mounted onto
a impermeable carrier. Put the affinity membrane with the coated
face towards the gel onto the gel, cover the backside of the mem-
brane with several layers of dry filter paper, wrap the stack into
a water-tight foil, and store it under moderate pressure in a refrig-
erator for several hours or overnight.
Open the sandwich, remove filter paper and gel, wash the mem-
brane several times with TBS or PBS, and incubate with a (labeled)
antibody, generated in another species and directed against a sec-
ond epitope. Wash again and detect the second antibody by the
usual procedures.
2.5.9 Transfer of Nucleic Acids (S
OUTHERN
and Northern Blot)
The transfer protocols are the same for DNA and RNA. In opposi-
tion to the methods given in Protocol 2.5.3, the forces driving the
biomolecules from the separating gel to the receiving membrane
are diffusion and capillary flow. This type of transfer is also ap-
plicable for proteins, but because the pores of polyacrylamide gels
used for protein separation are mostly smaller, transfer times are
longer and transfer efficiencies are lower than by electrotransfer.
Transfer (blotting) of DNA to membranes was introduced by
E.M. Southern in 1974, and as a pun the transfer of RNA as the
opposite of DNA was named Northern blot and that of proteins got
the direction West.
A . 5N hydrochloric acid
Solutions/Reagents
B .6M NaCl in 0.2 N NaOH
C .5M NaCl in 0.5 M Tr i s
HCl, pH 7.0
D3M NaCl in 0.3 M sodium citrate buffer, pH 8 (“20
·
×
SSC”)
Agitate the nondenaturated DNA gel (cf. Protocol 2.2.1) carefully
in its tenfold volume of Soln. A at RT for 30 min.Skipthisstepif
the DNA is significantly smaller than 5000 bp.
Decant the hydrochloric acid, add ten gel volumes of Soln. B,
and shake for additional 30 min, repeating this step with Soln. C
instead of Soln. B.
Place a glass plate somewhat larger than the gel over a tray
filled with Soln. D. Wet thick filter paper, e.g., Whatman 3MM,
with Soln. D and spread it over the glass plate, the ends dipping
into Soln. D. Now place the gel on the filter paper and remove air
bubbles. Cover the sides of the gel by strips of plastic wrap and put
the wetted membrane onto the gel.
