Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 19)

Section 8
Solutions for Protein Applications
8.1 Solutions for Nitrocellulose Membrane
Tris-Buffered Saline, 1x TBS, 2 L
20 mM Tris, pH 7.5
500 mM NaCl
Dissolve 4.84 g Tris, 58.48 g NaCl in ddH
2
O. Adjust to pH 7.5 with HCl.
Adjust the volume to 2 L with ddH
2
O.
Tween 20 Wash Solution, 1x TTBS, 1 L
20 mM Tris, pH 7.5
500 mM NaCl
0.05% Tween 20
Add 0.5 ml Tween 20 to 1 L of TBS.
Blocking Solution, 100 ml
3% gelatin-TBS
Add 3.0 g gelatin to 100 ml TBS. Heat at 37°C to dissolve the gelatin.
Antibody Buffer, 200 ml
1% gelatin-TTBS
Add 2 g gelatin to 200 ml TTBS. Heat at 37°C to dissolve the gelatin. 100 ml should be reserved
for primary antibody and an equal volume for dilution of the secondary antibody conjugate.
Primary Antibody Solution, 100 ml
Dilute antigen specific primary antibody to the appropriate titer in 100 ml of antibody buffer.
Secondary Antibody Solution, 100 ml
Dilute Bio-Rad’s species-specific secondary antibody conjugate, 1:3,000 by adding 33 µl of
conjugate to 100 ml of antibody buffer.
Color Development Solution
The specific chemicals and buffers are dependent on the enzyme conjugate being used. See the
Immun-Blot
® assay kit instruction manual for details on how to make the appropriate solution.
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