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Agglutinated

sarcolemma

SL vesicles

Highly enriched sarcolemma

Agglutinated SL vesicles

+purified WGA lectin

WGA

SL

+0.01% Triton X-100

2 min at 15 000 g

2 min at 15 000 g

20 min at 150 000 g

SN

Agglutinated and

solubilised SL

vesicles

SN

Deagglutinated SL vesicles

+0.2 M NAG

Supernatant

Crude surface membrane

Mixture of sarcolemma, transverse tubules and sarcoplasmic reticulum

(a)

(b)

600

400

100

30

Relative molecular weight

standards (

×^10

–3)

123 123 123

Total protein SL marker Non-SL marker

Gel/blot lane 1: Crude surface membrane

Gel/blot lane 2: Lectin void fraction

Gel/blot lane 3: Highly purified sarcolemma

WGA

WGA

WGA

Scheme of subcellular fractionation of muscle sarcolemma

Diagram of immunoblot analysis of subcellular fractionation procedures

Fig. 3.6Affinity separation method using centrifugation of lectin-agglutinated surface membrane vesicles
from skeletal muscle. Shown is a flow chart of the various preparative steps in the isolation of highly
purified sarcolemma vesicles (a) and a diagram of the immunoblot analysis of this subcellular fractionation
procedure (b). The sarcolemma (SL) and non-SL markers are surface-associated dystrophin of 427 kDa and
the transverse-tubulara1S-subunit of the dihydropyridine receptor of 170 kDa, respectively.