ROTI®Blot 1

Buffers recommended for Tank blotting:
Acc. to Tobwin et al., 1997: 25 mM Tris, 192 mM glycin, 20 % methanol. Optional: 0-0,1 % SDS.
Acc. to Bjerrum und Schaefer-Nielsen, 1986: 48 mM Tris, 39 mM glycin, 0-20 % methanol. Optional: 0-0.1 % SDS.
Acc. to Dunn, 1986: 10 mM NaHCO₃, 3 mM NaCO₃, 20 % methanol.

Buffers recommended for Semy Dry blotting:
Discontinuous buffer system: ROTI^®Blot 1 - for standard proteins or ROTI^®Blot 2 - for hydrophobic proteins.
Continuous Tris-glycin buffer acc. to Bjerrum and Schaefer-Nielsen, 1986: 48 mM Tris, 39 mM glycin, 0-20 % methanol. Optional: 0,01-0,1 % (usually 0,0375 %) SDS.
Continuous CAPS buffer for blotting of basic proteins and prior to N-terminal sequencing: 0,22 % CAPS (pH 10,5-11,0), 10 % methanol.
Do not adjust pH of the buffer (exception: CAPS stock solution). Adding acid or base to the buffer will result in higher conductivity, leading to increased formation of heat as well as ions. Subsequently, this results in brown, “burnt” looking blotting paper and damage to the electrode plates.

Methanol prevents the gel from swelling during transfer (mandatory for gradient gels!) and improves the absorption of proteins to NC membranes. For transfer of native or big proteins, reduce or omit methanol.

SDS improves transfer efficiency of large proteins and protein binding to PVDF membranes, however increasing relative current, power and temperature. Don’t use SDS for Semi-Dry blotting of small proteins to NC membranes. For tank blotting use 0,02 % SDS in minimum.

For blotting of native proteins use buffer acc to Bjerrum and Schaefer-Nielsen with 0,04 % SDS and 0-10 % methanol.