The myb gene family has three members, c-myb, A-myb, and B-myb, which have distinct expression patterns. Analyses of c-myb and A-myb mutant mice have indicated that c-myb and A-myb are important for hematopoiesis and spermatogenesis, respectively. However, there has been no evidence for a role for B-myb in development. To examine the role of B-myb in development, we generated B-myb-deficient mice by gene targeting. Although the heterozygous mutants were healthy, the homozygous mutants died at an early stage of development, around E4.5-E6.5. In vitro culture of blastocyst indicated that B-myb is required for inner cell mass formation. Consistent with the important role of B-myb in early embryonic development, only B-myb among myb family members was expressed in embryonic stem cells. These results indicate that each of the three members of the myb gene family plays a distinct role during development.

A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5-10 min with 0.1-0.2% colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42,650) in 10% w/v phosphoric acid. After staining for only 0.5-3 min, major zones, corresponding to 100-500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3% w/v phosphoric acid for 5-80 min depending on gel thickness (120-500 microns) and type of support (fabric reinforced versus gels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1-2 ng/mm2 protein is found. Dye elution from stained fabric reinforced gels with 50% v/v dioxane-water, followed by absorbance measurements, results in a linear relationship over a range of 1-100 micrograms marker proteins. Staining with collodial Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the collodial dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazardous.

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.

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