Bio-Rad Trans-Blot® Cell - Manual (Page 18)

Table 5.1 Guide to Protein Blotting Membranes
A variety of blotting membranes is available for immunoblotting, each with particular advan-
tages depending on the needs of the experiment. The physical properties and performance char-
acteristics of a membrane should be evaluated in selecting the appropriate transfer conditions.
Binding
Capacity
Membrane Pore Size (µg/cm 2 ) Notes
Nitrocellulose 0.45 µm 80–100 General purpose protein blotting membrane
0.2 µm
Supported 0.45 µm 80–100 Pure nitrocellulose cast on an inert synthetic support;
Nitrocellulose 0.2 µm increased strength for easier handling and for reprobing.
PVDF 0.2 µm 170–200 High mechanical strength and chemical stability, used for
protein sequencing, and western blotting; enhanced binding
in the presence of SDS. Must be wet in alcohol before equi-
libration in buffer.
Nylon 0.2 µm 170 Recommended for nucleic acids.
Note: Nucleic acids cannot be transferred to nitrocellulose by electrophoretic blotting.
Use Zeta-Probe membrane.
Section 6
Troubleshooting Guide
Electrophoretic Transfer
Poor electrophoretic transfer (as detected by staining the gel)—Proteins
1. Transfer time is too short.
• Increase the transfer time.
2. 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 appli-
cations in Section 3.
• Remake the buffer or increase the voltage.
• Try the high intensity blotting option.
3. 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 facing the opposite orientation. Check
the polarity of the connections to the power supply.
4. Charge-to-mass ratio is incorrect.
• 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. Protein is precipitating in the gel.
• Try using SDS in the transfer buffer. SDS can increase transfer efficiency,
but can also reduce binding efficiency to nitrocellulose and affect reac-
tivity of some proteins with antibodies.
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