Bio-Rad Trans-Blot® Plus Cell - Manual (Page 27)

Section 6
Troubleshooting Guide
Poor transfer of proteins
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 may be inserted in the tank with the wrong orientation. Check the
polarity of the connections to the power supply.
2. Detection system is not working or is not sensitive enough.
• Include proper positive and negative control antigen lanes to test for kit
sensitivity. Consult kit manual.
• Stain the gel after transfer with a total protein stain, like Coomassie ® Blue
or SYPRO Ruby, to make sure that proteins have left the gel.
3. Transfer time is too short.
• Increase the transfer time.
4. Power is too low.
• Always check the current at the beginning of the run. The current may be
too low for a particular voltage setting. If the buffer is prepared improperly,
the conductivity may be too low, and not enough power will be delivered
to the cell. See the power guidelines for specific applications in Section 3.
• Prepare new buffer or increase the voltage.
• Try the high intensity blotting option.
5. 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 will transfer poorly. (Buffer pH should
be 2 pH units higher or lower than the pI of the protein of interest for
optimal transfer efficiency.)
6. 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.
7. 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 membranes.
Protein is precipitating in the gel
1. Use SDS in the transfer buffer. SDS can increase transfer efficiency, but it can
also reduce binding efficiency to nitrocellulose and affect reactivity of some
proteins with antibodies.
2. Eliminate alcohol from the transfer buffer (see Section 4).
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