IEF and 2-D Standards

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Overview

IEF Standards

IEF standards allow dependable and reproducible pI calibration in IEF gels. IEF standards consist of a mixture of nine native proteins with isoelectric points ranging from 4.45 to 9.6. Five of the nine proteins are naturally colored to provide continuous monitoring of the focusing process. IEF standards are for use in analytical acrylamide and agarose isoelectric focusing gels under native conditions. The mixture is provided in a stable aqueous solution, and no reconstitution or dilution is required prior to use.


Constituent Proteins of IEF Standards*
Protein Color pI
1. Cytochrome c
Red
9.6
2. Lentil lectin (3 bands)
7.8, 8.0, 8.2
3. Human hemoglobin C
Red
7.5
4. Human hemoglobin A
Red
7.1
5. Equine myoglobin (2 bands)
Brown
6.8, 7.0
6. Human carbonic anhydrase
6.5
7. Bovine carbonic anhydrase
6.0
8. β-lactoglobulin B
5.1
9. Phycocyanin (3 bands)
Blue
4.45, 4.65, 4.75

* Because the IEF standards are in native form, they cannot be used with reducing or denaturing agents such as urea, β-mercaptoethanol, or dithiothreitol. For calibration or IEF tube gels containing urea, use 2-D SDS-PAGE standards.

IEF standards strained with IEF gel stain. Run on Criterion™ (left) and Ready Gel™ IEF gels (right).

IEF standards for accurate pI calibration of native IEF gels.
A. 5 µl of the standards were stained with Coomassie Blue R-250 dye and crocein scarlet
B. 5 µl of the standards were left unstained

2-D SDS-PAGE Standards

These unique protein standards calibrate the pI and molecular weight of the proteins in 2-D SDS-PAGE applications. The standards consist of seven reduced, denatured proteins that can be run internally on the same gel as the sample, or alone to test the ampholyte mixture and the reproducibility of the gels. Protein spots can be visualized with silver or Coomassie Blue staining. No dilution of the standards is required.

Two-dimensional electrophoretic protein pattern of 2-D SDS-PAGE standards

Two-dimensional electrophoretic protein pattern of 2-D SDS-PAGE standards. The standards (2.5 μl) were run on 7 cm ReadyStrip™ IPG strips, then in the Mini-PROTEAN® II cell. For method details, see Klose (1975), Klose and Feller (1981), and Jungblut and Seifert (1990). The gel was stained with Bio-Rad’s silver stain kit.

IEF Standards

IEF standards allow dependable and reproducible pI calibration in native polyacrylamide or agarose IEF gels. IEF standards are a mixture of nine native proteins with pI ranging from 4.45 to 9.6. To monitor focusing, five of the nine proteins are naturally colored. The standards are provided in a stable aqueous solution, and need no reconstitution or dilution prior to use.

Constituent Proteins of IEF Standards*

Protein Color pl
1. Cytochrome c Red 9.6
2. Lentil lectin (3 bands) 7.8, 8.0, 8.2
3. Human hemoglobin C Red 7.5
4. Human hemoglobin A Red 7.1
5. Equine myoglobin (2 bands) Brown 6.8, 7.0
6. Human carbonic anhydrase 6.5
7. Bovine carbonic anhydrase 6.0
8. β-Lactoglobulin B 5.1
9. Phycocyanin (3 bands) Blue 4.45, 4.65, 4.75

* Because the IEF standards are in native form, they cannot be used with reducing or denaturing agents such as urea, β-mercaptoethanol, or dithiothreitol. For calibration of IEF tube gels containing urea, use 2-D SDS-PAGE standards.

2-D SDS-PAGE Standards

These unique protein standards provide calibrated references for the pI and molecular weight of proteins in 2-D SDS-PAGE applications. They consist of seven reduced, denatured proteins that can be visualized with silver or Coomassie Blue staining. No dilution is required.

Constituent Proteins of 2–D SDS-PAGE Standards

Protein pl MW
1. Hen egg white conalbumin  6.0, 6.3, 6.6 76,000
2. BSA  5.4, 5.5, 5.6 (empirically determined) 66,000
3. Bovine muscle actin 5.0, 5.1 (empirically determined) 43,000
4. Rabbit muscle GAPDH
8.3, 8.5 36,000
5. Bovine carbonic anhydrase  5.9, 6.0 31,000
6. Soybean trypsin inhibitor  4.5 21,500
7. Equine myoglobin
7.0 17,500

MW size range Varies by standard
Stain type Unstained
Number of applications Depends on staining method
Volume IEF: 250 µl; 2D: 500 µl
Shelf life 12 months at –20°C
IEF Standards

161-0310
250 μl, pI range: 4.45–9.6, phycocyanin, β-lactoglobulin B, bovine and human CA, equine myoglobin, human hemoglobin A and C, lentil lectin, and cyt C

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2-D SDS-PAGE Standards

161-0320
500 μl, 17,500–76,000 MW range, pI 4.5–8.5, unstained mixture of myoglobin, trypsin inhibitor, CA, GAPDH, actin, BSA, and conalbumin

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Number Description Options
2414 The Little Book of Standards, Rev D Click to download [ Add to Cart (Free) ]
3118 Protein Standards for Electrophoresis and Blotting Brochure, Rev D Click to download [ Add to Cart (Free) ]
2998 Protein Standards Application Guide, Rev G Click to download [ Add to Cart (Free) ]
LIT396 Instruction Manual, IEF Standards, Rev F Click to download
4110064 Instruction Manual, 2-D SDS-PAGE Standards, Rev A Click to download