Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

Patent · Sun Jan 01 00:00:00 EST 1989

OSTI ID:866928

Zhang, Jian-Shi ^[1]; Giometti, Carol S ^[2]; Tollaksen, Sandra L ^[3]

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1. Shanghai, CN
2. Glenview, IL
3. Montgomery, IL

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

• Research Organization: Argonne National Laboratory (ANL), Argonne, IL (United States)
• DOE Contract Number: W-31109-ENG-38
• Assignee: United States of America as represented by United States (Washington, DC)
• Patent Number(s): US 4824547
• OSTI ID: 866928
• Country of Publication: United States
• Language: English

Related Subjects

electrophoretic
extraction
proteins
two-dimensional
electrophoresis
spots
resulting
polyacrylamide
slab
pattern
protein
thereon
individual
spot
cored
form
core
placed
tube
dialysis
membrane
replicate
slabs
molten
agarose
poured
hardens
immobilizing
covering
cores
upper
portion
filled
volume
buffer
solution
closed
bodies
brought
contact
membranes
provided
electrodes
connected
positive
negative
terminals
dc
power
supply
producing
electrical
current
flows
causes
extracted
electrophoretically
accumulate
contained
space
percentage
achieved
removed
subjected
partial
digestion
trypsin
followed
peptide
extract
peptides
electrodes connected
dc power
power supply
electrical current
buffer solution
two-dimensional electrophoresis
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