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
Section 4
Instructions for Use
Preparation of Protein Solubilization Buffer (PSB)
Solution
1. Use only freshly rehydrated buffer. Discard any unused
buffer.
2. Allow the PSB dry reagent to warm to room
temperature before opening the bottle. Shake the PSB
dry reagent bottle for 10–15 sec. Weigh an appropriate
amount (each gram of dry reagent will prepare
approximately 2 ml buffer solution). Use 1 ml of PSB
per 60 µl of wet cell pellet (Table 1).
Table 1. Guideline for PSB preparation.
# Samples Volume PSB PSB Dry PSB Diluent Approximate
(60 µl wet cell Needed (ml) Reagent (g) (ml) Volume PSB
pellet) Prepared (ml)
1 1 1 1.1 2
2 2 2 2.2 4
3 3 2 2.2 4
4 4 3 3.3 6
5 5 3 3.3 6
3. For each gram of dry reagent, add 1.1 ml of PSB
diluent.
4. Vortex periodically and incubate at room temperature
until you have a clear solution (2–3 min).
4
5. Add reducing agents, protease inhibitors, and carrier
ampholyte as needed (Table 2).
Table 2. Additions to PSB solution recommended
for various applications. Note that though the
applications listed often require use of chaotropes and
detergents, these agents are already included in the PSB
solution.
Protein
Component Extraction IEF Separation
MicroRotofor Cell IPG Strip
Carrier ampholyte NA 2% (w/v) 0.2% (w/v)
DTT* 50–100 mM 50–100 mM 50–100 mM
or
TBP* 2–5 mM 2–5 mM 2–5 mM
Protease inhibitor According to NA NA
manufacturer
Bromophenol Blue NA NA 0.002% (w/v)
Glycerol NA 10% NA
*Not needed if reduction-alkylation is performed at step 17.
Sample Processing
6. Suspend the wet yeast cell pellet (~60 µl) in 100 µl of
yeast suspension buffer.
Note: For best results, use a wet cell pellet from a freshly
grown yeast culture.
5
7. Add 1 µl of β-mercaptoethanol per 100 µl of yeast
suspension buffer.
8. Vortex until the cell suspension becomes homogenous.
9. Flick the vial of lyticase enzyme to mix. Add 10 µl of
lyticase enzyme per 100 µl of yeast cell pellet. Gently
mix. Lyticase will hydrolyze poly-(beta-1,3-glucose) for
lysis of the yeast cell wall.
10. Incubate the cell suspension at 37°C for 30–60 min.
11. Centrifuge the suspension at 10,000 x g for 5 min.
Remove and discard the supernatant carefully, leaving
the spheroplast pellet in the tube.
Note: If the majority of the sample is mucous-like and
difficult to pipet, the spheroplasts may have lysed. Reduce
the incubation time or start with a fresh culture.
12. Optional wash step: Add 600 µl of yeast suspension
buffer to the spheroplast pellet. Resuspend the
spheroplasts by gently tapping the tube. Centrifuge
again as above and discard the supernatant.
Note: This step washes away the lyticase from the protein
sample. Including this wash step may compromise the
yeast protein yield. Should you choose to eliminate this
step, you may anticipate the migration of lyticase on a 2-D
gel by knowing its pI (6.33) and molecular weight (54.6 kD).
6
13. Add 600 µl of freshly prepared PSB solution to the
spheroplast pellet.
14. Sonicate the suspension to break down the cell
membrane and the genomic DNA. Sonication should
be performed in an ice bath to prevent heating.
Sonication should be performed with bursts of
20–30 sec, with chilling of the suspension on ice
between bursts.
15. Centrifuge at 20,000 x g for 30 min at 20°C and collect
the clear lysate.
16. Resuspend the residual cell debris in 250 µl of PSB
solution. Sonicate the suspension once briefly. Repeat
the centrifugation in step 15, collect the supernatant,
and pool with the first supernatant.
17. Determine the protein concentration of the extract.
This is best done using the RC DC protein assay
(catalog #500-0121 or 500-0122), which is compatible
with the detergents and reducing agents in PSB. If
performing the RC DC protein assay, keep in mind that
two washes of the sample are recommended. (Optional:
A reduction and alkylation of the sample is recommended
at this point in the procedure. Refer to the ReadyPrep
reduction-alkylation kit, catalog #163-2090.) Store the
protein extract at –70°C, apply directly onto an IPG
strip (see Appendix for recommendations), or proceed
to step 18.
7
Preparing Extracts for a MicroRotofor Run (See
Section 6 of MicroRotofor manual for alternative sample
preparation and load conditions.)
18. Prepare fresh PSB solution containing PSB diluent,
glycerol, carrier ampholyte, and DTT or TBP (DTT or
TBP is not required if a reduction-alkylation step is
performed at step 17). See Table 2 for recommendations.
19. One MicroRotofor run requires ~2.5 mg protein
(1 µg/µl) in a total volume of 2.5 ml. Using the above
prepared PSB solution, prepare 2.5 ml of a 1 µg/µl
dilution of the protein extract. Load the entire 2.5 ml
sample into the MicroRotofor chamber. It may be
necessary to add extra PSB solution to fill the chamber
completely, eliminating any void volumes.
20. Run the MicroRotofor cell according to the
MicroRotofor instruction manual.
Note: Following fractionaction with the MicroRotofor cell it
is recommended to perform an SDS-PAGE analysis profiling
all 10 fractions. This will illustrate the protein content of
each fraction. See the Appendix for recommendations
pertaining to SDS-PAGE analysis of MicroRotofor fractions.
For subsequent analysis of MicroRotofor fractions by 2-D
PAGE, the ampholyte concentration in samples should not
exceed 0.2–0.5%. If fractions contain high amounts of protein,
dilution prior to loading onto the IPG strip (by 1:10 or greater)
8
will be sufficient to reduce the ampholyte concentration. In
cases where protein levels are lower, use of the ReadyPrep
2-D cleanup kit (catalog #163-2130) for ampholyte removal
is recommended.
Section 5
Appendix
Preparation for SDS-PAGE
CHAPS, a component of the PSB diluent, may interfere
with SDS-PAGE. Remove CHAPS from the extracts (for
example, with the ReadyPrep 2-D cleanup kit) or dilute the
extracts 1:1 with 1x Laemmli buffer prior to SDS-PAGE.
Preparation for IEF on an IPG Strip
Following step 17, the sample extract can be loaded onto
an IPG strip after appropriate dilution. See Table 3 for
recommendations on how much protein sample to load
onto an IPG strip. Dilution of the sample can be done using
protein solubilization buffer (PSB) as a rehydration/sample
buffer. However, some critical components need to be added
to the PSB solution to make it IEF-compatible (Table 2).
9
Table 3. Recommended protein loads for IPG
strips.
IIPPGG SSttrriipp LLeennggtthh
77 ccmm 1111 ccmm 1177 ccmm 1188 ccmm 2244 ccmm
Rehydration volume/strip 125 µl 185 µl 300 µl 315 µl 410 µl
Protein load
Silver stain 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg
Coomassie G-250 50–100 µ g 100–200 µg 200–400 µg 200–400 µg 400–800 µg
Flamingo™,
SYPRO Ruby 2.5–75 µg 10–150 µg 25–300 µg 25–300 µg 40–600 µg
The suggestions made in Table 3 are a general rule of
thumb. Increased protein loads may be required for
micro-range IPG strips and for samples of higher protein
complexity.
Lyticase Enzyme
This enzyme is prepared from Arthrobacter luteus. The
primary yeast lytic activity is beta1, 3-glucan
laminaripentaohydrolase, which hydrolyzes glucose
polymers at the beta-1,3-glucan linkages, releasing
laminaripentaose as the principal product.
Unit definition: One lytic unit is defined as a 10% decrease
in absorbance at A
800
in 30 min.
Assay condition: 50 mM potassium phosphate, pH 7.5,
10 mM β-mercaptoethanol in 1 ml yeast cell suspension of
A
800
0.8 to 1.0.
10
Store at –20°C when frequently used. Store below –70°C
for infrequent usage (less than once a month). Lyticase is
stable for 1 year at –20°C and for many years below
–70°C.
Section 6
References
Harbers, A et al., Fractionation by liquid-phase isoelectric focusing in the
MicroRotofor cell: improved detection of low-abundance proteins, Bio-Rad
bulletin 5344 (2005)
Scott J et al., Lyticase: Endoglucosnase and protease activities that act
together in yeast cell lysis, J Bacteriol 142, 414–423 (1980)
Vuillard L et al., Non-detergent sulfobetaines: a new class of mild
solubilization agents for protein purification, Biochem J 305, 337–343 (1995)
11
Section 7
Product Information
Catalog # Description
Sample Preparation Kits
163-2141 MicroRotofor Lysis Kit (Mammal)
163-2142 MicroRotofor Lysis Kit (Plant)
163-2143 MicroRotofor Lysis Kit (Yeast)
163-2144 MicroRotofor Lysis Kit (Bacteria)
163-2145 Protein Solubilization Buffer (PSB)
163-2146 ReadyPrep Mini Grinders, 20 tubes with
resin and pestles
163-2130 ReadyPrep 2-D Cleanup Kit, 50 preps
163-2140 ReadyPrep 2-D Cleanup Kit, 5 preps
163-2090 ReadyPrep Reduction-Alkylation Kit,
100 preps
170-2836 MicroRotofor Syringes, 3 ml and
10 ml, 3 each
Protein Quantitation Kits (see also bulletin 2610)
500-0121 RC DC Protein Assay Kit I, 500 standard
assays, bovine γ-globulin standard
500-0122 RC DC Protein Assay Kit II, 500 standard
assays, bovine serum albumin standard
12
Buffer Components
161-0611 Dithiothreitol (DTT), 5 g
163-2101 Tributylphosphine (TBP), 200 mM, 0.6 ml
163-2091 ReadyPrep Proteomic Grade Water, 500 ml
163-2094 Bio-Lyte
®
3/10 Ampholyte, 100x, 1 ml
161-0737 Laemmli Sample Buffer, 1x, 30 ml
Coomassie is a trademark of BASF Aktiengessellschaft. SYPRO is a
trademark of Molecular Probes, Inc.
13
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
(510) 741-1000
1-800-424-6723
10005509 Rev A
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