Protein Sample Preparation

Protocol

Bulletin 6194

General Tips
Keep the sample preparation workflow simple (increasing the number of sample handling steps may
increase variability).

Lysis (Cell Disruption) — If protein phosphorylation is to be studied, include

■■ 
Suspend ~1 mg (wet weight) pelleted cells in ~10 µl phosphatase inhibitors such as okadaic acid, calyculin A,
1× SDS-PAGE sample buffer for a protein concentration and vanadate.
of 3–5 µg/µl. If disrupted in liquid nitrogen, tissue samples ■■  hen working with a new sample, use at least two
W
like liver biopsies and plant leaves contain 10–20% and
different cell disruption protocols and compare the protein
1–2% protein, respectively
yield (by protein assay) and qualitative protein content
■■ To diminish endogenous enzymatic activity: (by SDS-PAGE)
— Disrupt the sample or place freshly disrupted samples ■■  ptimize the power settings of mechanical rupture systems
O
in solutions containing strong denaturing agents and incubation times for all lysis approaches. Mechanical
such as 7–9 M urea, 2 M thiourea, or 2% SDS. In this cell lysis usually generates heat, so employ cooling where
environment, enzymatic activity is often negligible required to avoid overheating of the sample
 — Perform cell disruption at low temperatures to diminish ■■  ollowing cell disruption, check the efficacy of cell wall
F
enzymatic activitiy disruption by light microscopy and centrifuge all extracts
extensively (20,000 × g for 15 min at 15°C) to remove any
 — Lyse samples at pH >9 using either sodium carbonate or
insoluble material; solid particles may block the pores of the
Tris as a buffering agent in the lysis solution (proteases
electrophoresis gel
are often least active at basic pH)
 — Add a chemical protease inhibitor to the lysis buffer.
Examples include phenylmethylsulfonylfluoride
(PMSF), aminoethylbenzylsulfonylfluoride
(AEBSF), tosyllysinechloromethylketone
(TLCK), tosylphenylchloromethyletone (TPCK),
ethylenediaminetetraacetic acid (EDTA), benzamidine,
and peptide protease inhibitors (for example, leupeptin,
pepstatin, aprotinin, bestatin). For best results, use a
combination of inhibitors in a protease inhibitor cocktail.
Protein Solubilization Preparation for PAGE
■■ 
Solubilize proteins in a buffer that is compatible with the ■■ 
Prepare SDS-PAGE sample buffer without reducing agent,
corresponding electrophoresis technique then aliquot and store it at –70°C
■■ Dissolve pelleted protein samples in 1× sample buffer ■■  repare reducing agent fresh, and add it to SDS-PAGE
P
■■  ilute dissolved protein samples with sample buffer stock
D sample buffer immediately before use
solutions to a final sample buffer concentration of 1× ■■  issolve dry protein samples directly in 1× sample buffer;
D
■■  erform a protein quantitation assay to determine the
P prepare other protein sample such that the final sample
amount of total protein in each sample. Use a protein assay buffer concentration is 1×
that is tolerant to chemicals in your samples. For samples in ■■ Incubate samples in sample buffer at 95°C for 5 min
Laemmli sample buffer, for example, use the DC or RC DC (or at 70°C for 10 min) after addition of sample buffer for
protein assays, which can tolerate up to 10% detergent more complete disruption of molecular interactions
■■  ilute or concentrate samples as needed to yield a final
D ■■  hen preparing SDS-PAGE sample buffer, use either
W
protein concentration of >0.5 mg/ml (omit the protein assay 5% (~100mM) 2-mercaptoethanol (bME) or 5–10 mM
if sample amount is limited) dithiothreitol (DTT)
■■  se protein extracts immediately or aliquot them into
U ■■  referably, the final protein concentration in the sample
P
appropriately sized batches and store them at –70°C to solution for 1-D electrophoresis should not be <0.5 mg/ml.
avoid freeze-thaw cycles Keep in mind that the final sample concentration should
■■  or long-term sample storage, store aliquots at –70°C;
F also be consistent with the sensitivity of your detection
avoid repeated thawing and freezing of protein samples method as well as with the complexity of the sample.
Higher sensitivity detections can be used with lower sample
■■  ighly viscous samples likely have a very high DNA or
H
concentrations. In general, the more complex samples
carbohydrate content. Fragment DNA with ultrasonic waves
require higher sample concentration for analysis
during protein solubilization or by adding endonucleases
like Benzonase. Use protein precipitation with TCA/acetone
(for example, with the ReadyPrep 2-D cleanup kit) to
diminish carbohydrate content
■■  hen a sample preparation protocol calls for a dilution,
W
the two parts are stated like a ratio, but what is needed is
a fraction. For example, “Dilute 1:2,” means to take 1 part
of one reagent and mix with 1 part of another, essentially
diluting the part by one half. “Dilute 1:4,” means to take
1 part and mix with 3 parts, making a total of 4 parts,
diluting the part by a quarter.

This is an excerpt from Bio-Rad’s comprehensive Electrophoresis Guide (Bulletin 6640).

Bio-Rad
Laboratories, Inc.

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Bulletin 6194 Rev A US/EG 11-0864 1111 Sig 1211