Invitrogen iBlot 2, Руководство пользователя

Страница: 46 / 68

Observation Possible cause Recommended action
Difficulty closing lid.
Note: Do not press forcibly on
the lid as it can cause damage
to the unit.
Transfer stack was too thick. The iBlot
™
2 Gel Transfer Device can only
support gels of ≤1.5mm thickness. Ensure the
proper gel is being used.
Ensure that additional filter pads have not been
added. Only use the supplied filter paper with
the iBlot
™
2 Transfer Stacks.
Ensure the stack tray is properly aligned. Do
not remove the transfer stack from the plastic
sample tray.
No proteins transferred to the
membrane.
No current or incorrect method
used.
See previous page to ensure the electrical
circuit is complete and current is flowing
through the device. Be sure to use the correct
Method (“Recommended running
parameters“ on page 23).
Empty spots on the membrane. Presence of air bubbles
between the gel and the
membrane prevented the
transfer of proteins.
Be sure to remove all air bubbles between the
gel and the membrane using the Blotting
Roller.
Expired or creased membranes
were used.
Use the iBlot
™
2 Transfer Stacks before the
expiration date printed on the package.
High molecular weight proteins
remain in the gel indicated by
staining of the gel after
transfer.
Incorrect method or transfer
conditions used.
Note: It is normal for some
proteins to remain in the gel
because some high molecular
weight proteins do not transfer
completely using the iBlot
™
2
Gel Transfer Device, compared
to semi-wet transfer apparatus.
Use the appropriate method and run time
based on the gel type as described
“Recommended running parameters“ on
page 23.
For mini or midi gels:
• Perform ethanol equilibration step as
described “Optimizing blotting“ on
page 49 to improve transfer.
• Use a Tris-acetate gel to separate the high
molecular weight proteins.
• Increase the transfer time in 30 ‑ second
increments.
For E-PAGE ™ gels:
• Increase the transfer time in 30 ‑ second
increments.
• Use Method P3 for 8 minutes.
Protein blow-through. Transfer time was too long. Reduce transfer time by 30-second
increments.
Note: Pre-stained markers are charged and
tend to blow-through more than regular
proteins.
Protein bands distorted on
membrane.
Non-uniform electric field
created around wells.
Ensure the gel is properly flattened using the
Blotting Roller. Follow the recommendations
on page 20 to obtain good results.
Appendix A Troubleshooting
Introduction
A
46 iBlot
™
2 Dry Blotting System User Guide