Manual

Section 2
Protocol
2.1 Experimental Strategy and General
Considerations
Temperature. All steps are performed at room
temperature (22–25 °C).
Making Solutions. Use only deionized, distilled water
to prepare solutions. 1x PBST solutions should be sterile fil-
tered. Do not use azide as a preservative in any solution.
Membrane Selection. This kit has been optimized
for detection on pure nitrocellulose membranes and per-
forms equally well with supported nitrocellulose mem-
branes. However, due to the way in which they are
manufactured, the surface of some supported nitrocellulose
membranes take on a ‘wavy’ appearance that can result in
less pleasing images than the pure nitrocellulose mem-
branes which remain flat throughout the process. The kit
may also be used to detect proteins bound to PVDF.
Primary Antibody. Generally when serum or tissue
culture supernatants are the source of primary antibody, a
1:100–1:1,000 dilution of the primary antibody in buffer
is used for detection of antigens on the membrane sur-
face. For chromatographically purified monospecific anti-
bodies, a 1:500–1:10,000 dilution is used for antigen
detection. A 1:1,000–1:100,000 dilution is used when
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Antibodies are provided in 10 mM sodium phos-
phate, 150 mM sodium chloride, pH 7.2, with 1% (w/v)
bovine serum albumin and 0.01% thimerosal (sodium
ethylmercurithiosalicylate) as a preservative. Avoid
freeze-thaw cycles of antibody conjugates which will
cause breakdown of product. For best results, aliquot
conjugates in appropriate amounts and store at -20 °C.
1.5 Safety Instructions
Please read the entire instruction manual before
beginning the protocol.
1. Wear gloves and protective clothing, such as labora-
tory coats and goggles when preparing and working
with the solutions in the protocol. DMSO is an irritant;
it is a colorless liquid which is easily absorbed through
the skin and mucous membranes. Avoid skin contact
with DMSO and inhalation of DMSO mist. Wash
exposed skin thoroughly with soap and water.
Note: See Material Data Safety Sheet (MSDS) on DMSO
for additional information.
2. Work in well-ventilated areas. Avoid inhalation of
vapors when working with solutions containing DMSO.
3. Do not mouth pipet any solution.
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