Thermo Scientific OWL HEP-1, Руководство по эксплуатации и техническому обслуживанию

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Semidry Electroblotter 4-1 Thermo Scientific
Section 4
Troubleshooting
Problem Cause Solution
Transfer efficiency is poor • Current is too low
• Semidry transfer should be performed at constant
current. Current density should be between 0.5 and
3mA/cm2 of stack surface area.
• Power supply is inappropriate for semidry
transfer
• Many power supplies will shut off or blow a fuse
when run at the conditions required for semidry
transfer. Semidry transfer requires low voltage (often
less than 10V) and high current. Check with the man-
ufacturer of the power supply to determine whether
it is appropriate for semidry transfer.
• Transfer performed for too short a time • Increase the amount of time for transfer.
• Transfer sandwich was assembled in the
wrong order
• The semi-dry electroblotter is configured with the
cathode on the bottom, and anode on top. This
means that an upward transfer is being performed
rather than downward. Follow the instructions care-
fully when assembling the transfer sandwich.
• The pH of the transfer buffer is too close
to the isoelectric point of the protein
• Try a more acidic or basic transfer buffer.
• Too much methanol in the transfer buffer
• Reducing methanol can help elute proteins from
the gel, but can reduce binding to nitrocellulose
membranes.
• High percentage gels restrict transfer
• Higher percentage acrylamide or crosslinker can
restrict elution of proteins. Use the lowest percent-
age acrylamide possible to separate your proteins.
• Puddles of buffer were present on the
cathode allowing the current to bypass the
stack
• Always clean up the lower plate before closing
the lid of the transfer apparatus. Do not squeeze the
stack excessively as this also creates puddles that
the current can pass through.
• The filter paper was too dry
• Filter paper should be saturated with transfer
buffer before adding them to the sandwich.