(http://www.ebiotrade.com/)

NOTE:

For a listing of available cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, and attachment agents, see General Laboratory Support Products.

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Prepare a total cell lysate as described under Western blot procedure in protocol 1.

NOTE:

For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology, Inc.磗 PhosphoCruz?Protein Purification System (sc-24964).

?/FONT>	Preclear whole cell lysate (optional step) as follows. To approximately 1 ml of whole cell lysate or tissue extract (see whole cell lysates or tissue extract tables), add 0.25 礸 of the appropriate control IgG (corresponding to the host species of the primary antibody; see Control IgGs and IgG Conjugates), together with 20 祃 of appropriate suspended (25% v/v) agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Incubate at 4?C for 30 minutes.
?/FONT>	Pellet beads by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4?C. Transfer supernatant (cell lysate) to a new microcentrifuge tube at 4?C.
?/FONT>	To 1 ml of the above cell lysate, or approximately 100?000 礸 of total cellular protein, add 10 礸 of primary antibody agarose conjugate (i.e., 5 祃 volume of packed beads) and incubate at 4?C for 1 hour to overnight with mixing.
?/FONT>	Alternatively, if primary antibody agarose conjugate is not available, incubate 1 ml cell lysate with 1?0 祃 (i.e., 0.2? 礸) primary antibody (optimal antibody concentration should be determined by titration) for 1? hours at 4?C. Add 20 祃 of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose or Protein L-Agarose). Cap tubes and incubate at 4?C on a rocker platform or rotating device for 1 hour to overnight.
?/FONT>	Collect pellet by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4?C. Carefully aspirate and discard supernatant.
?/FONT>	Wash pellet 2? times with either RIPA buffer (sc-24948) (more stringent) or PBS (Buffers and General Solutions) (less stringent), each time repeating centrifugation step above.
?/FONT>	After final wash, aspirate and discard supernatant and resuspend pellet in 40 祃 of 2x electrophoresis sample buffer (sc-24945).
?/FONT>	Boil samples for 2? minutes. Load up to 5?0 祃 of sample per 1.0 mm well width for gels of 0.75 mm thickness.
?/FONT>	Continue with electrophoresis and immunoblotting as described under Western blotting procedure in protocol 1.

NOTE:

Depending on the secondary antibody that is used, 55 kDa and 27 kDa heavy and light IgG chains, respectively, of the primary antibody may be detected. These bands will be less pronounced if a primary antibody agarose conjugate is used in the above procedure or if ExactaCruz?Reagents are used.

Support Products

Cruz Marker?Compatible Secondary Antibodies for Western Blotting

Cruz Marker?Molecular Weight Standards

Western Blotting Chemiluminescene Luminol Reagent

Prestained Molecular Weight Standards

Western Blotting Membranes

Blocking Reagents for Western Blotting Applications

Cruz Blot?Systems for Western Blotting

Conventional Secondary Antibodies for Western Blottiing

Isotype-Specific Secondary Antibodies for Western Blotting

Adult Tissue Extracts for Western Blottiing

Embryonic and Postnatal Tissue Extracts for Western Blotting

Immunoprecipitation Reagents

Whole Cell Lysates for Immunoprecipitation

Control IgGs and IgG Conjugates

Purification Reagents

GST-Agarose Preclearing Reagent

Nuclear Extracts for Gel Shift and Western Blotting

PhosphosCruz&trade Protein Purification System

Phospho-Enriched Tissue Extracts for Immunoprecipitation

Phospho-Enriched Whole Cell Lysates for Immunoprecipitation

Tissue Extracts for Immunoprecipitation

Acetylation Specific Antibodies

Western Blotting Supplies

General Laboratory Support Products