Bio-Rad PROTEAN® IEF Cell Accessories - Manual (Page 2)

Specifications
Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
IPG Strip Dimensions
IPG strip length, cm
7.9 11.8 17.8 19.0 24.7
Gel length, cm 7.3 11.0 17.1 18.0 23.4
Strip width, mm 3.3 3.3 3.3 3.3 3.3
Linear pH Gradient Range
Broad
range 3–10 3–10 3–10 3–10 3–10
3–10 NL* 3–10 NL* 3–10 NL* 3–10 NL* 3–10 NL*
Narrow range 3–6, 4–7, 3–6, 4–7, 3–6, 4–7, 3–6, 4–7, 3–6, 4–7,
5–8, 7–10 5–8, 7–10 5–8, 7–10 5–8, 7–10 5–8, 7–10
Micro range 3.9–5.1, 3.9–5.1, 3.9–5.1, 3.9–5.1, 3.9–5.1,
4.7–5.9, 4.7–5.9, 4.7–5.9, 4.7–5.9, 4.7–5.9,
5.5–6.7, 5.5–6.7, 5.5–6.7, 5.5–6.7, 5.5–6.7,
6.3–8.3 6.3–8.3 6.3–8.3 6.3–8.3 6.3–8.3
When rehydrated, IPG strips will be approximately 0.5 mm thick. The gel
composition for each IPG strip is 4%T/3%C. The anode (acidic) end of each IPG
strip is indicated with a “+” symbol. Storage temperature is –20ºC. Each package
contains 12 IPG strips.
* NL, Nonlinear gradient.
Introduction
The combination of isoelectric focusing (IEF) with SDS-PAGE
provides a powerful technique for the separation of complex
mixtures of proteins. ReadyStrip IPG strips are made with buffering
acrylamide derivatives that contain either a free carboxylic acid or
a tertiary amino group, which is copolymerized with acrylamide
and bis-acrylamide. As such, the pH gradient is precast into
the gel and cannot shift during electrophoresis. The precast
IPG strips provide reproducible gradients in an easy-to-use
format. The ReadyPrep
™ 2-D starter kit (catalog #163-2105) is
recommended for performing 2-D electrophoresis using IPG strips
or for performing troubleshooting and method development. More
detailed information on 2-D techniques and specific information
about related products can be found in the 2-D Electrophoresis
Manual for Proteomics (bulletin 2651) as well as product-specific
instruction manuals.
Rehydration volumes
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Volume 125 µl 200 µl 300 µl 315 µl 450 µl
12 hours is the recommended rehydration time.
Solutions
Rehydration buffer
Standard Method
Optimization Guidelines
8 M urea 7–9.8 M urea, 0–2 M thiourea*
2% CHAPS 1–4% CHAPS*
50 mM dithiothreitol (DTT) 15–100 mM DTT*
0.2% Bio-Lyte ® ampholytes or 1x ReadyStrip buffer 0.1–0.4% (w/v)
Bio-Lyte ampholytes**
* The amounts of urea, CHAPS, DTT, and Bio-Lyte ampholytes required depend
on the sample solubility. The amounts listed here serve as a general guideline.
The optimal rehydration solution composition for each sample type is best
determined empirically.
Recommended carrier ampholyte mixtures by IPG strip pH range
IPG Strip
Buffer IPG Strip Buffer
3–10 Bio-Lyte 3/10 7–10 ReadyStrip 7–10 buffer*
3–10 NL Bio-Lyte 3/10 3.9–5.1 ReadyStrip 3.9–5.1 buffer*
4–7 Bio-Lyte 3/10 4.7–5.9 ReadyStrip 4.7–5.9 buffer*
3–6 Bio-Lyte 3/10 5.5–6.7 ReadyStrip 5.5–6.7 buffer*
5–8 Bio-Lyte 3/10 6.3–8.3 ReadyStrip 6.3–8.3 buffer*
* When 100x ReadyStrip buffers are diluted to 1x, the final sample yields a
concentration of 0.2% Bio-Lyte ampholytes.
Recommended protein load ranges
IPG Strip Length 7 cm
11 cm 17 cm 18 cm 24 cm
Protein Load
Staining Method
Silver stain
5–20 μg 20–50 μg 50–80 μg 50–80 μg 80–150 μg
Coomassie G-250 50–100 μg 100–200 μg 200–400 μg 200–400 μg 400–800 μg
Fluorescent stain
5–100 μg 20–200 μg 50–400 μg 50–400 μg 80–800 μg
Focusing Conditions
Broad and narrow ranges
Start Voltage Find Voltage Volt-Hours Ramp Temperature
ReadyStrip pH 3–10, 3–10 NL, 4–7, 5–8*
7 cm
0 V 4,000 V 8–15,000 V-hr Rapid 20°C
11 cm 0 V 8,000 V 20–35,000 V-hr Rapid 20°C
17 cm and
18 cm 0 V 10,000 V 40–60,000 V-hr Rapid 20°C
24 cm 0 V 10,000 V 60–80,000 V-hr Rapid 20°C
ReadyStrip pH 3–6 Focusing Conditions** , ***
7 cm 0 V 4,000 V 8–10,000 V-hr Rapid 20°C
11 cm 0 V 8,000 V 15–20,000 V-hr Rapid 20°C
17 cm and
18 cm 0 V 10,000 V 30–40,000 V-hr Rapid 20°C
24 cm 0 V 10,000 V 40–55,000 V-hr Rapid 20°C
ReadyStrip pH 7–10* , †
7 cm 0 V 4,000 V 8–16,000 V-hr Rapid 20°C
11 cm 0 V 8,000 V 20–30,000 V-hr Rapid 20°C
17 cm and
18 cm 0 V 10,000 V 40–50,000 V-hr Rapid 20°C
24 cm 0 V 10,000 V 60–70,000 V-hr Rapid 20°C
Micro ranges
Start Voltage End Voltage Set Time Ramp Temperature
ReadyStrip pH 3.9–5.1, and 4.7–5.9***
Step 1
7 cm
0 V 250 V 15 min Rapid 20°C
11 cm 0 V 250 V 20 min Rapid 20°C
17 cm and
18 cm 0 V 250 V 30 min Rapid 20°C
24 cm 0 V 250 V 30 min Rapid 20°C
Step 2
7 cm
250 V 4,000 V 1 hr Gradual 20°C
11 cm 250 V 8,000 V 1 hr Gradual 20°C
17 cm and
18 cm 250 V 10,000 V 2 hr Gradual 20°C
24 cm 250 V 10,000 V 2 hr Gradual 20°C
Step 3
7 cm
4,000 V 4,000 V 10–20,000 V-hr Rapid 20°C
11 cm 8,000 V 8,000 V 20–32,000 V-hr Rapid 20°C
17 cm and
18 cm 10,000 V 10,000 V 30–50,000 V-hr Rapid 20°C
24 cm 10,000 V 10,000 V 60–90,000 V-hr Rapid 20°C
ReadyStrip pH 5.5–6.7, and 6.3–8.3 †
Step 1
7 cm
0 V 250 V 15 min Rapid 20°C
11 cm 0 V 250 V 20 min Rapid 20°C
17 cm and
18 cm 0 V 250 V 30 min Rapid 20°C
24 cm 0 V 250 V 30 min Rapid 20°C
Step 2
7 cm
250 V 4,000 V 1 hr Gradual 20°C
11 cm 250 V 8,000 V 1 hr Gradual 20°C
17 cm and
18 cm 250 V 10,000 V 2 hr Gradual 20°C
24 cm 250 V 10,000 V 2 hr Gradual 20°C
Step 3
7 cm
4,000 V 4,000 V 20–30,000 V-hr Rapid 20°C
11 cm 8,000 V 8,000 V 30–45,000 V-hr Rapid 20°C
17 cm and
18 cm 10,000 V 10,000 V 45–63,000 V-hr Rapid 20°C
24 cm 10,000 V 10,000 V 60–90,000 V-hr Rapid 20°C