Preparing POS fragments

Department of Physics, Willamette University

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Preparing photoreceptor outer segment (POS) fragments

1) Re-suspend 10 POS in 5 mL of 10 mM HEPES

• We have 1 M HEPES stock (pH 7.3)

• Made 10 mM stock from 1 M frozen stock: 5 mL 1 M HEPES, raise to 500 mL and sterile filter

2) Pass 30 times through 20-gauge canula – in the cold room

3) Aliquot into 100 uL stocks

4) Assess protein concentration (Bradford assay)

• From communciations with Linda Bapst, they measure ~1.4 ug/uL

• We measured 450-650 ug/uL (standard curve was a little noisy)

• Freeze in -80 for long term storage