Bio-Rad Criterion™ Blotter User Manual
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Section 6
Troubleshooting Guide
6.1 Electrophoretic Transfer
Poor or no electrophoretic transfer (as detected by staining the gel)
1. Transfer apparatus is assembled incorrectly, and the proteins are moving in the wrong
direction.
•
The gel/membrane sandwich may be assembled in the wrong order or the cassette is
inserted in the tank with the incorrect orientation. Check the polarity of the
connections to the power supply.
2. Detection system is not working or not sensitive enough.
•
Include proper positive and negative control antigen lanes to test for detection kit
sensitivity. Consult kit manual.
3. Transfer time is too short.
•
Increase the transfer time.
4. Charge-to-mass ratio is incorrect (Native transfers).
•
Try a more basic or acidic transfer buffer to increase protein mobility. Proteins near
their isoelectric point at the pH of the buffer will transfer poorly. (It has been
suggested that buffer pH should be 2 pH units higher or lower than the pI of the
protein of interest for optimal transfer efficiency.)
5). Power supply circuit is inoperative, or an inappropriate power supply was used.
•
Check the fuse. Be sure the voltage and current output of the power supply match the
needs of the blotting instrument.
6. Methanol in the transfer buffer is restricting elution.
•
Reduction of methanol results in increased transfer efficiency of proteins from the gel,
but it also diminishes binding to nitrocellulose and PVDF.
Protein is precipitating in the gel
1. Try using SDS in the transfer buffer. SDS can increase transfer efficiency, but can also
reduce binding efficiency to nitrocellulose and affect reactivity of proteins with some
monoclonal antibodies.
Swirls or missing bands; diffuse transfers
1. Poor contact between the membrane and the gel. Air bubbles or excess buffer remain
between the blot and gel.
•
Use the included roller, test tube, or pipet as a rolling pin, and roll over the
membrane carefully in both directions until air bubbles or excess buffer is removed
from between gel and membrane, and complete contact is established.
•
Use thicker filter paper in the gel/membrane sandwich.
•
Replace the fiber pads. Pads will compress with time, and will not hold the
membrane to the gel.
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