376
ROBERT F. REILLY
3. Wash each lane with 1 ml of BLOTTO five times at 10 min intervals.
4. Add a 1:1000 dilution of secondary antibody in 1 ml of BLOTTO to each
lane and incubate with constant agitation for 1 hr. The secondary antibody employed will depend on both the species of animal in which the
primary antibodywas generated, and the detection system used. Wehave
used two enzymatic detection systems (alkaline phosphatase (15) and
horseradish peroxidase ). If using a horseradish peroxidase detection system BLOTTO without azide should be used from this point onward since
azide inhibits this enzyme.
5. W ash again with BLOTTO as in step 3.
6. For alkaline phosphatase-conjugated secondary antibodies, wash with 1
ml of substrate buffer (0.1 M NaCl, 0.1 M Tris, 5 mM MgClz, (pH 9.5)). To
develop add 500 J..ll of substrate (ratio is 4 mls ofwater, 0.5 ml of5-bromo4-chloro-3-indoyl phosphate concentrate, and 0.5 ml nitro blue tetrazolium concentrate) to each lane. Allow the reaction to proceed. Stop the
reaction by rinsing with distilled water when desired. The substrate can
be obtained from Kirkegaard and Perry Laboratories, 2 Cessna Court,
Gaithersburg, MD, USA.
7. Wehave developed horseradish peroxidase-conjugated secondary antihodies with the ECL Western blotting detection system as recommended
by the manufacturer (Amersham). We currently use this system exclusively. Its advantages compared to the alkaline phosphatase detection
system include: greater sensitivity; transfers carried out using Immobilon-P( can be stripped and reprobed (16); exposure time can be varied to
obtain a better image.
References
1. Shih D, and Kaesberg P (1976) Translation ofthe RNAs ofbrome mosiac virus: the
monocystronic nature of RNA1 and RNA2. J Mol Biol103: 77-88
2. Morch MD, Drugeon G, Zagorski W, and Haenni AL (1986) The synthesis ofhighmolecular-weight proteins in the wheat germ translation system. Meth in Enzymol
118: 154-164
3. Pelham HRB, and Jackson RJ (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Bioehern 67: 247-256
4. Adamson SD, Herbert E, and Godschaux W (1968) Factors affectingthe rate ofprotein synthesis in lysate systems from reticulocytes. Arch Bioehern Biophys 125: 671683
ROBERT F. REILLY
3. Wash each lane with 1 ml of BLOTTO five times at 10 min intervals.
4. Add a 1:1000 dilution of secondary antibody in 1 ml of BLOTTO to each
lane and incubate with constant agitation for 1 hr. The secondary antibody employed will depend on both the species of animal in which the
primary antibodywas generated, and the detection system used. Wehave
used two enzymatic detection systems (alkaline phosphatase (15) and
horseradish peroxidase ). If using a horseradish peroxidase detection system BLOTTO without azide should be used from this point onward since
azide inhibits this enzyme.
5. W ash again with BLOTTO as in step 3.
6. For alkaline phosphatase-conjugated secondary antibodies, wash with 1
ml of substrate buffer (0.1 M NaCl, 0.1 M Tris, 5 mM MgClz, (pH 9.5)). To
develop add 500 J..ll of substrate (ratio is 4 mls ofwater, 0.5 ml of5-bromo4-chloro-3-indoyl phosphate concentrate, and 0.5 ml nitro blue tetrazolium concentrate) to each lane. Allow the reaction to proceed. Stop the
reaction by rinsing with distilled water when desired. The substrate can
be obtained from Kirkegaard and Perry Laboratories, 2 Cessna Court,
Gaithersburg, MD, USA.
7. Wehave developed horseradish peroxidase-conjugated secondary antihodies with the ECL Western blotting detection system as recommended
by the manufacturer (Amersham). We currently use this system exclusively. Its advantages compared to the alkaline phosphatase detection
system include: greater sensitivity; transfers carried out using Immobilon-P( can be stripped and reprobed (16); exposure time can be varied to
obtain a better image.
References
1. Shih D, and Kaesberg P (1976) Translation ofthe RNAs ofbrome mosiac virus: the
monocystronic nature of RNA1 and RNA2. J Mol Biol103: 77-88
2. Morch MD, Drugeon G, Zagorski W, and Haenni AL (1986) The synthesis ofhighmolecular-weight proteins in the wheat germ translation system. Meth in Enzymol
118: 154-164
3. Pelham HRB, and Jackson RJ (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Bioehern 67: 247-256
4. Adamson SD, Herbert E, and Godschaux W (1968) Factors affectingthe rate ofprotein synthesis in lysate systems from reticulocytes. Arch Bioehern Biophys 125: 671683