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Troubleshooting Hoefer DALT System
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Amersham Biosciences
Poor Spot Resolution
• Allow gel to polymerize fully.
• Begin electrophoresis as soon as the IPG strips are loaded to prevent low
molecular weight species from diffusing.
• Conduct the separation at a lower current or voltage setting.
• Reduce the temperature setting
Reagent quality and gel preparation
• Use only the highest quality reagents.
• Only use gels that were recently prepared.
• Check pH values of the stacking gel solutions. Do not back-titrate buffers.
Sample preparation
• Store IPG strips at –40 °C or below.
• Add a protease inhibitor, such as phenylmethylsulfonyl fluoride ( PMSF), if
necessary, to prevent proteolytic degradation of sample.
Insufficient equilibration
• Extend equilibration to 15 minutes.
• Add DTT and iodoacetamide fresh before use.
Tracking Dye Doesn’t Sharpen into a Concentrated Zone in the Stacking Gel
• Dispose of outdated acrylamide solutions and use only the highest grade of
acrylamide.
• Buffer reused too many times. Prepare fresh each time, for best results.
Incomplete Transfer
Blank areas on the membrane
• Remove all trapped air pockets in the transfer stack assembly. Assemble
the stack while it is submerged in transfer buffer. Gently press on each
sponge as it is added to the stack. Roll a glass pipette or test tube over the
membranes and gel to eliminate all bubbles.
• Process only one strip or membrane in each tray or cassette to prevent
overlapping.
• Use buffer with a lower ionic strength.
• Check electrode continuity. During the transfer, a continuous stream of gas
is released along the entire length of the electrodes. If bubbles do not form
along the entire length of the electrode, replace the electrode.
Grid pattern on membrane
• Add sheets of blotting paper to increase the clearance between the
cassette panel and the gel. Take care not to overstuff the cassette. The gel
should be held firmly and evenly between the sponges, but not so tightly
that it is squeezed.
Molecules do not migrate out of gel
• Increase the field strength.
• Increase, or double, the transfer period.
• Do not use staining or fixing agents on the gel before transfer.
• Use a thinner gel.
• Reduce the gel acrylamide concentration.
• Check that the buffer pH is close to the intended pH. Most buffers should
not be titrated. Make fresh buffer.
• Use 3.5 mM SDS (0.1%) in the transfer buffer.
• Use reagent-grade chemicals.
• Increase the net charge on the protein by changing to a transfer buffer with
a different pH. Lower pH (<6) increases the positive charge on proteins.
Higher pH (>6) increases the negative charge on proteins.