ISSN:
0173-0835
Keywords:
Density gradient electrophoresis
;
Endosomes
;
Clathrin
;
Proteasomes
;
Proteins
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
A density gradient electrophoresis (DGE) apparatus (2.2 Ø, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 μS/cm buffer, and basic proteins in a pH 5.4, 76 μS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of β-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 μS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 μS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrincoated vesicles within a single run directly from a postnuclear supernatant.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150190718
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