Bio-Rad Criterion™ Blotter - Manual (Page 8)

2.3 Acidic Transfers
If transferring under acidic buffer pH, switch the gel and membrane in the set up
instructions or simply reverse the orientation of the gel assembly or reverse the cassette when
inserted in to the tank. This will place the membrane on the cathode side of the gel. Under
acidic conditions, proteins will transfer in the opposite direction going toward the negative
cathode. Do not reverse the electrodes themselves, or plug the banana plugs into the
reverse poles. This will cause irreversible damage to the plate electrodes.
Section 3
Transfer Conditions
3.1 General Guidelines to Transfer Buffers and Running
Conditions
Tables 3.1 to 3.4 provide guidelines for power conditions using different buffers. Power
conditions are provided for various run times. The transfer times will need to be increased
for gradient gels, or they may be decreased if your protein of interest is low molecular weight
and transfers quickly. The suggested conditions give at least 95% transfer of the sample
proteins visible in the gel by Silver Stain Plus, catalog number 161-0449 (sensitive to ng
level) and in the blot by Colloidal Gold, catalog number 170-6517 (sensitive to 4 ng).
Table 3.1 SDS-PAGE Gels
These conditions were determined empirically using 12.5% Tris-HCl Criterion gels and total
proteins from E.coli lysates.
Buffer: 1X Tris/Glycine (see section 3.3 Buffer formulation)
20% Methanol 10% Methanol 15% Ethanol
Criterion Blotter 100 V 100 V Not recommended *
with plate electrodes 30 minutes 30 minutes
Criterion Blotter 100 V 100 V Not recommended *
with wire electrodes 60 minutes 30 minutes
* Our tests show only 60% transfer of E.Coli proteins in 1 hour at 100V. The ethanol buffer might work if longer transfers are
acceptable or if your target protein transfers under this condition.
Table 3.2 SDS PAGE Gels (CAPS based buffers)
These conditions were determined empirically using 12.5% Tris-HCl Criterion gels and total
proteins from E.coli lysates.
Buffer: 10mM CAPS buffer (see Section 3.3 Buffer formulation)
20% Methanol 10% Methanol 15% Ethanol
Criterion Blotter 100 V 100 V 100 V
with plate electrodes 30 minutes ** 30 minutes ** 30 minutes
Criterion Blotter 100 V 100 V 100 V
with wire electrodes 60 minutes ** 30 minutes ** 60 minutes
** We find nearly undetectable levels of proteins remaining in the gel, but some protein blow through is observed under this
condition. PVDF is suggested for transfer of low molecular weight proteins.
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