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A single polyacrylamide gel was run this week and was transferred to nitrocellulose. The proteins on the membrane were probed with Streptavidin-AP (at a dilution of 1:20,000) to detect biotin-labeled Ltv1-His. Fractions sampled were

• the mixture of in vitro translation products and nonspecific BL21 proteins ("input"),
• the flow-through (supernatant) from the GST-Yar1 beads after input was added ("Yar1 flow-through"),
• he GST-Yar1 beads after washing and boiling in sample buffer ("Yar1 beads"),
• the flow-through from the GST control beads ("GST flow-through"),
• and the GST beads after washing and boiling in sample buffer.

Kaleidoscope marker proteins were loaded as molecular size standards. Their migration is marked with () on the left side of each exposure. The four smallest markers indicated on each blot are (from bottom up) 33 kDa, 42 kDa, 80 kDa, and 116 kDa.

A five-minute exposure of the membrane to X-ray film gave the following results:

A longer exposure was performed to determine whether any labeled protein could be detected in the "Yar1 beads" lane. 17 minutes of exposure to film gave the following results:

As before, the presumptive Ltv1-His band is migrating slightly slower than the 80 kDa marker band. There is no detectable Ltv1-His on the GST-Yar1 beads. This is disappointing, but not too surprising given our finding last week that much of the GST-Yar1 dissociated from these beads during the incubation with in vitro product. A potentially larger concern is that there may be a small amount of Ltv1-His on the (negative) control GST beads--it's difficult to tell from this blot whether there is a band there or not.

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