Bio-Rad MicroRotofor Cell Lysis Kits User Manual

MicroRotofor™Lysis Kit
(Yeast)

Instruction Manual

Catalog #163-2143

For technical support, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)

Table of Contents

Section 1 Introduction....................................................1

Section 2 Kit Specifications ..........................................1

Section 3 Storage Conditions ........................................3

Section 4 Instructions for Use........................................4

Section 5 Appendix ......................................................9

Section 6 References ..................................................11

Section 7 Product Information ....................................12

Section 1
Introduction

MicroRotofor lysis kits provide convenient, effective methods
for the preparation of protein samples for fractionation with
the MicroRotofor cell. The MicroRotofor lysis kit (yeast) is
designed for use with yeast cultures, and employs enzymatic
digestion of the cell wall (Scott et al. 1980) followed by
solubilization into a chaotropic extraction buffer (Vuillard
et al. 1995). For added convenience, the extraction buffer
is also used as the sample buffer for isoelectric focusing
(IEF) either with the MicroRotofor cell or with IPG strips.

Section 2
Kit Specifications

Each MicroRotofor lysis kit (yeast) provides sufficient
reagent to perform at least 15 extractions (from 5 ml yeast
culture with OD

600

= 1.48, which should yield a 60 µl wet
cell pellet) and to prepare sample for 15 MicroRotofor runs.
More than 15 extractions will be possible with the kit if the
sample is applied onto IPG strips (and not prefractionated
with the MicroRotofor cell).

Each MicroRotofor run using 2.5 mg total protein yields ten
150–250 µl fractions, and the protein distribution among

1

the fractions will vary depending on the sample. For example,
using Saccharomyces cerevisiae and ampholytes spanning
the pH range 3–10, fractions 2–4 typically contain the most
protein.

Certificates of analysis and MSDS forms are available upon
request.

Items Supplied With the Kit

Protein solubilization buffer (PSB) (contains urea, 25 g
thiourea, NDSB 201, and Tris)

PSB diluent (contains CHAPS and Tris) 30 ml

Yeast suspension buffer (contains sodium phosphate, 15 ml
sodium chloride, potassium chloride, and
potassium phosphate)

Lyticase enzyme, 5 units/µl, prepared from Arthrobacter 2 x 0.5 ml
luteus, MW = 54.6 kD, pI = 6.33 Swiss-Prot/TrEMBL
accession number E13B-ARTSW (Q59146). See Appendix for
more information

Instruction manual 1

Items Required But Not Provided

• 1.5 ml microcentrifuge tubes

• Microcentrifuge capable of spinning at 20,000 x g

• Sonicator with probe

• DTT reducing agent (catalog #161-0611) or TBP reducing

agent (catalog #163-2101)

• Carrier ampholytes

• RC DC

™

protein assay (catalog #500-0121 or 500-0122)

• Glycerol

2

• ReadyPrep™proteomic grade water (catalog #163-2091)

or other ultrapure water

• β-mercaptoethanol

Items Recommended But Not Required

• Protease inhibitor (for example, Sigma catalog #P8215)

• ReadyPrep reduction-alkylation kit (catalog #163-2090)

• ReadyPrep 2-D cleanup kit (catalog #163-2130)

Section 3
Storage Conditions

Shipped at ambient temperature. Store kit components as
individually marked. Note: Lyticase is shipped at room
temperature, but should be stored at –20°C upon receipt.
This kit has a warranty period of 1 year from shipment
date, assuming all components are stored as indicated on
each label.

Component Store at

Protein solubilization buffer (PSB), 25 g RT
PSB diluent, 30 ml 4°C
Yeast suspension buffer, 15 ml RT
Lyticase, 1.5 U/µl, 2 x 0.5 ml –20°C

3