Bio-Rad PROTEAN® IEF Cell Accessories - Manual (Page 3)

By plotting the pH of an IPG strip as a function of its length, the pI of a protein
may be derived from its focused position on that strip. A, the pH vs. length
relationship of a linear pH 4–7 IPG strip, indicating how to estimate the pI of a protein
that migrates to 50% of the gel length; B, the pH vs. length relationship of a nonlinear
pH 3–10 IPG strip.
A. Linear pH 4–7 ReadyStrip IPG strip
pH
7
6
5
4
0 25 50 75 100
B. Nonlinear pH 3–10 ReadyStrip IPG strip
pH
10
9
8
7
6
5
4
3
0 25 50 75 100
Total IPG strip length, %
Second dimension electrophoresis
IPG Strip
Length 7 cm 11 cm 17 cm 18 cm 24 cm
Electrophoresis Mini-PROTEAN ® Criterion ™ PROTEAN II XL PROTEAN PROTEAN
cells Tetra, PROTEAN ® and Plus Plus
Dodeca Criterion Dodeca Dodeca
Dodeca
Electrophoresis 200 V,
200 V, 16 mA/gel for 200 V, 200 V,
conditions constant constant 30 min, then constant constant
24 mA/gel for
~5 hr
Approximate
15–25 min 30–55 min 5.5 hr 6–8 hr 6–8 hr
run time
Recommended equilibration volumes
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Equilibration buffer I 2.5 ml 4 ml 6 ml 6 ml 8 ml
Equilibration buffer II 2.5 ml 4 ml 6 ml 6 ml 8 ml
10 minutes is recommended for each equilibration step.
Bar Coding
The 24 cm IPG strip contains bar coding to assist in high-
throughput applications. The automated bar code provides a
unique 6-character identifier for each IPG strip. The recommended
bar code reader is the Gryphon L GD 4330 laser scanner
(Part # GD4330-BKK1).* Within the bar coding area there is a
human readable portion for convenient information management
without the use of a bar code reader. The figure below illustrates
the human-readable and automated bar coding regions.
* Infinite Corporate Solutions, Inc. 1400 Battleground Ave., Suite 207-B Greensboro,
NC 27408, USA. Tel. 866-271-6556.
Estimating the pI of a protein from
its position along an IPG strip
Part Numbers
ReadyStrip IPG strips, 12 per package
pH Range
7 cm 11 cm 17 cm 18 cm 24 cm
3–10 163-2000 163-2014 163-2007 163-2032 163-2042
3–10 NL* 163-2002 163-2016 163-2009 163-2033 163-2043
4–7 163-2001 163-2015 163-2008 163-2034 163-2044
3–6 163-2003 163-2017 163-2010 163-2035 163-2045
5–8 163-2004 163-2018 163-2011 163-2036 163-2046
7–10 163-2005 163-2019 163-2012 163-2037 163-2047
3.9–5.1 163-2028 163-2024 163-2020 163-2038 163-2048
4.7–5.9 163-2029 163-2025 163-2021 163-2039 163-2049
5.5–6.7 163-2030 163-2026 163-2022 163-2040 163-2050
6.3–8.3 163-2031 163-2027 163-2023 163-2041 163-2051
* NL, Nonlinear gradient.
* The final voltage for each pH range may not be reached, but the total volt-hours
given above are sufficient to properly focus samples with final voltages as low
as 3,000 V (7 cm), 5,000 V (11 cm), and 7,000 V (17 cm, 18 cm, and 24 cm).
A lower final voltage will increase total run time.
** The final voltage for this pH range may not be reached, but the total volt-hours
given above are sufficient to properly focus samples with final voltages as low
as 2,000 V (7 cm), 3,000 V (11 cm), and 6,000 V (17 cm, 18 cm, and 24 cm).
A lower final voltage will increase total run time.
*** Enhanced resolution and separation of proteins may be achieved using cup
loading with sample application at the cathode (–) end of the IPG strip.
† To ensure success with basic range IPG strips, performing two additional steps is
strongly recommended. The first step is to treat the sample using the ReadyPrep
reduction-alkylation kit (catalog #163-2090). This reduces streaking caused by
disulfide bond formation, which is more problematic with basic range proteins.
The second step is to use cup loading when loading samples for isoelectric
focusing. For more information, refer to the ReadyPrep reduction-alkylation kit
instruction manual (bulletin 4110063).