Two variants of lamin B3 are expressed in
Xenopus oocytes. Three nuclei each of noninjected
oocytes (A) or oocytes injected with RNA encoding lamin Flag-B3a (B) or
Flag-B3b (C) were manually isolated, and proteins were separated by IEF
in the first dimension (IEF) and by SDS-PAGE in the second dimension
(SDS). Lamins were detected after immunoblotting with
the use of either mAb L6-5D5, which recognizes lamin B3 [B3 (GV)], or
mAb M2, which recognizes the Flag epitope (Flag-B3a and Flag-B3b). The
shift in isoelectric point of the two epitope-tagged lamins, which is
due to the presence of the Flag epitope, is indicated by broken lines.
Note the higher resolution of the IEF gel in the range between pH 5.7
and 5.8 caused by the nonlinearity of the pH gradient. Isoelectric
points (averages) of standard proteins in the first dimension (left to
right: phosphorylase b, BSA, and ovalbumin) are given at
the top. The positions of the isoelectric variants of BSA and ovalbumin
are indicated by brackets within each blot. The sizes of molecular mass
markers in the second dimension are given in kilodaltons at the
right.