Protein Protocols Protein Protocols

Western Cross-Blotting 1029
3.2. Electrotransfer onto NC
1. For each gel, cut one sheet of NC paper and 15 pieces of Whatman 3MM paper to fit the
dimensions of the separating gel (see Note 6).
2. For each gel, prewet six sheets of the Whatman paper in buffer A, another six sheets in
buffer C, and three sheets in buffer B. Prewet the single sheet of NC paper in buffer B.
3. Disassemble the electrophoresis unit, discard the stacking gel, and mount the blot in the
following order, making sure that no air bubbles are trapped between individual stacks:
a. Place the Whatman stack from buffer A onto the anode plate of the semidry blotting
apparatus.
b. Place the Whatman stack from buffer B on top and cover it with the NC sheet.
c. Place the gel onto the NC paper, then cover it with the Whatman stack from buffer C.
4. Close the apparatus by mounting the cathode plate on top of the assembly and perform
electrophoresis at a constant current of 1.4 mA/cm 2 for 2 h.
3.3. Blocking and Incubation of the Blots with Antiserum
All incubations are done on a laboratory shaker, either at room temperature for the
time indicated or at 4°C overnight.
1. Disassemble blotting apparatus and incubate the blot, which is intended as the antibody
source (i.e., the “donor” blot) in PBS-Tween at room temperature for 30 min. Incubate the
second blot (“receptor” blot) in 1% BSA in PBS for 2 h.
2. Incubate the donor blot with antiserum in PBS-Tween with 1 mg/mL of BSA (see Note 7).
3. From the central area of both blots, cut square-shaped pieces fitted to the dimension of the
separating gel. Make asymmetrical marks on edges of the square-shaped NC pieces to
help remember the orientation of the antigen bands. Set aside the remaining pieces of the
donor blot in PBS-Tween. Incubate the remaining pieces of the receptor blot with antiserum
in PBS-Tween with 1 mg/mL of BSA. These margin pieces will serve as reference
strips to identify spots on the cross blot.
4. Place donor blot onto a glass filter and wash with 20 mL of PTS in aliquots of approx 3 mL
under vacuum using a sidearm flask. Incubate donor blot in PTS for 10 min on a laboratory
shaker.
5. Repeat step 4 twice (see Note 8).
3.4. Cross Blot
3.4.1. Electrotransfer of Antibodies
1. Prewet one stack of Whatman paper, 1 cm thick and sufficiently sized to cover the donor
blot, in BT and a second, equally sized, stack in SBT.
2. Prewet a piece of dialysis membrane (with a pore size of approx 10 kDa) in BT, which is
sufficiently sized to wrap the donor blot (i.e., at least twice the area of the donor blot).
3. Assemble the cross-blot as follows (Fig. 1):
a. Place the Whatman stack soaked in BT onto the anode of the semidry blotting apparatus.
b. Place the dialysis membrane on top, in such a way that one half covers the Whatman
stack while the second half rests beside it on the anode plate.
c. Place the receptor blot, with the protein bands upside, onto the part of the dialysis
membrane, which covers the anodal Whatman stack.