Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 6)

2.2 Total Protein Detection Procedure
Before beginning, read through the entire procedure. The
following procedure is based on 100 ml of incubation solution,
which is sufficient to assay one 15 x 15 cm size membrane in a
20 x 20 cm incubation vessel. This volume should be adjusted for
the membrane surface area to be stained and the size of the incu-
bation vessel (see Section 1.4).
1. There are three methods by which proteins may be bound to
membranes:
a. Dot-blotting: Cut the nitrocellulose or nylon membrane to
the appropriate size ( e.g. 0.6–0.8 x 15–20 cm strips, 3 x 5 cm
rectangles, or 15 x 15 cm squares). If desired, draw a
1 x 1 cm grid on the membrane with pencil. Wet the mem-
brane by slowly placing it in PBS at a 45° angle. Remove
the membrane(s) from PBS and dry on filter paper for
5 minutes. Apply 1 to 2 µl of sample to each square using a
Hamilton syringe or a variable pipette. Displace the sample
to the tip of the syringe or pipette as a drop and touch it to
the surface of the membrane. If the protein sample is very
dilute, it is possible to apply successive doses at the same
spot, taking care to let spot dry completely before applying
the next dose. The membrane should be allowed to dry
completely (approximately 5 minutes) before continuing to
Step 2.
b. Electrophoretic blotting: Follow the protocol outlined in
the Trans-Blot ® instruction manual. If desired, cut the
membrane into the appropriate lanes containing resolved,
transferred proteins. Place membrane strips in the incuba-
tion tray and proceed to Step 2.
c. Microfiltration blotting: The Biotin-Blot protein detection
method can easily be adapted for use with Bio-Dot ® micro-
filtration apparatus (catalog number 170-6550). All appli-
cations and washes, except for color development, are
carried out in the apparatus.
2. Wash the membrane in 100 ml BT solution for 10 minutes. If
the membrane has been in a buffer containing amines, repeat
the wash 2 times, 10 minutes each.
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Section 2
Total Protein Detection
2.1 General Recommendations
1. Handling the membrane. Wear clean plastic gloves or use
clean forceps to avoid fingerprints on the membrane. Gloves
or forceps which have been in amine containing solution
should not be used (see 3 below).
2. Temperature. All steps are performed at room temperature
(22–25 °C).
3. Membrane preparation. Total protein detection is based on
the derivatization of the amino groups of proteins bound to
nitrocellulose or nylon membranes. Any amine containing
materials, such as traces of Tris, or glycine from transfer
buffer, decrease the sensitivity of the assay. If the membrane
has been in buffer containing amines, it should be rinsed in BT
solution at least 3 times, 10 minutes each, prior to beginning
total protein detection.
4. Incubation vessels. Incubation vessels made of plastic are
preferred, since avidin binds to glass even in the presence of
detergents. Siliconized glass is acceptable. All vessels should
be clean and free of proteins or other amine containing com-
pounds. Such contaminants may compete with membrane
bound proteins for the biotinylating reagent and may affect the
sensitivity of the assay.
5. Membrane incubation. Agitation with a rotating shaker or
reciprocating platform enhances incubation efficiency. If
neither is available, hand mixing every few minutes and
extended incubation periods will suffice.
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