Bio-Rad Criterion™ Blotter - Manual (Page 9)

Table 3.3 Native Gels
These conditions were determined empirically using 12.5% Tris-HCl Criterion gels and four
native protein samples: cytochrome C (pI 9.6), lentil lectin (pI 8.2, 8.0, 7.8), carbonic
anhydrous (pI 6.0) and glucose oxidase (pI 4.5).
Buffer: 1X Tris/Glycine (see Section 3.3 Buffer formulation)
The transfer of proteins from Native gels will depend on the size and pI of the protein
relative to the pH of the buffer used during transfer. If the pI of the protein is greater than the
pH of the transfer buffer, the protein will carry a positive charge and will travel toward the
negative electrode (cathode). The voltage suggested is a starting point. The transfer time will
need to be determined empirically for your protein of interest.
Criterion Blotter Overnight (12 hrs) 30 minutes
with plate electrodes Max 10 V 50 V
Max 50 mA 750–950 mA
Criterion Blotter Overnight (12 hrs) 60 minutes
with wire electrodes Max 10 V 50 V
Max 50 mA 300–500 mA
Note: The power supply should be set on these maximum settings. The actual power supply reading may be lower throughout the
run. These conditions are excellent for neutral proteins (pI~6.0), as we found at least 90% of the carbonic anydrous protein transferred
successfully.
Table 3.4 DNA and RNA
These conditions were determined empirically using 5% uniform TBE Criterion gels and the
low range Fluorescein labeled DNA standards (catalog number 170-3123).
Buffer: 1X TBE (see section 3.3 Buffer formulation)
Criterion Blotter Overnight (12 hrs) 30 minutes
with plate electrodes 10 V 50 V
100 mA 750–950 mA
Criterion Blotter Overnight (12 hrs) 60 minutes
with wire electrodes 20 V 50 V
100 mA 300–500 mA
Note: The power supply should be set on these maximum settings. The actual power supply reading may be lower throughout the
run.
3.2 Notes on Electrophoretic Transfer Conditions
1. Pre-equilibration of gels
All gels should be pre-equilibrated in transfer buffer prior to electrophoretic transfer (may
not be necessary for native gels and nucleic acid gels where transfer buffer is generally the
same as running buffer). Pre-equilibration will facilitate the removal of contaminating
electrophoresis buffer salts and neutralization salts. If the salts are not removed, they will
increase the conductivity of the transfer buffer and the amount of heat generated during
the transfer. Also, gels will shrink to various degrees depending on the acrylamide
percentage in methanol buffers. Equilibration allows the gel to adjust to its final size prior
to electrophoretic transfer.
If the isoelectric point of the protein is close to the pH of the buffer, the protein may not
leave the gel. If the isoelectric point of the protein is 2 pH units below the pH of the
buffer, the protein will be negatively charged and will migrate towards the positive
electrode (anode).
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