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FIRST DIMENSION GUIDELINES AND FORMATS
 IPG strips simplify first-dimension separations by immobilizing the pH gradient on an easy-to-handle support strip. The strips are available in a wide selection of pH gradients and lengths from 7 cm up to 24 cm. The length and pH gradient selected will depend on the complexity of the sample and the amount of protein resolution desired.
When two strips have the same pH range but differ in length - the ratio of the strip lengths gives the relative focusing power of the longer strip. Thus, compared to a 7 cm pH 3-10 strip, the 11 cm pH 3-10 strip has a relative focusing power of 11 cm/7 cm = 1.6 -> longer strips can resolve more proteins.
When two strips have the same length but differ in pH range - the ratio of the pH ranges (in number of pH units) gives the relative focusing power of the strip with the smaller range. Thus, compared to a 7 cm pH 3-10 strip, the 7 cm pH 5-8 strip has a relative focusing power of (7 pH units)/(3 pH units) = 2.3 -> narrow pH ranges can resolve more proteins within that pH range.
Staff at the CNRU Mass Spectrometry Core Facility will be happy to assist you in the selection of strip sizes and ranges to meet your needs. Sample will be loaded onto IEF strips using the following guidelines:
| | Strip Size | Silver stain | Coomassie | Maximum |
| BioRad | 11cm | 50-200 | ug 250-1000ug | 1 mg |
| BioRad | 17 cm | 100-300ug | 1-3 mg | 3 mg |
| Amersham | 11 cm | 5-40ug | 20-600 ug | 600ug |
| Amersham | 18 cm | 15-120ug | 75-1500 ug | 1.5 mg |
We treat pH range 6-11 a little differently. We add a special "DeStreak" reagent to the samples so the sample buffer cannot contain other reductants (i.e. no DTT). DeStreak is hard on strips so we use Amersham strips for these experiments. We can only load 150µL for either 11 or 18 cm strips.
DEALING WITH INTERFERING SUBSTANCES
- Salts should not be present at greater than 10mM. We can do a special type of sample loading if salt is present at slightly greater concentrations, up to 50mM; however, we can only load 150µL in this case. Spin columns, dialysis, filtration, or precipitation can be used to get rid of salts.
- Small molecules such as nucleotides, and lipids can be removed by precipitation. Nucleotides can be treated with nucleases (DNase, RNase, or endonuclease (Sigma))
- Ionic detergents, such as SDS, can either be diluted to less than 0.25%, or the sample can be precipitated.
The tables below provide the formats and part numbers of the isoelectric focusing strips we employ to separate proteins in the first dimension. Loading volumes and concentrations, as well as approximate focusing times are provided as a guideline. BioRad strips have different requirements than strips provided by GE Healthcare (Amersham).
BIORAD IPG STRIPS |
| Strip Size | pH 3-10 NL | pH 3-6 * |
pH 4-7 | pH 5-8 | pH 7-10 * |
11 cm
| 5 hr
163-2016 |
6.5 hr
163-2017 |
4 hr
163-2015 |
4 hr
163-2018 |
4 h
163-2019 |
| 17 cm |
7.5 hr
163-2009 |
9 hr
163-2010 |
6.5 hr
163-2008 |
6 hr
163-2011 |
6.5 hr
163-2012 |
| *Cup load |
| Loading vol | Total conc | Max conc | Pre-swelling |
| 11 cm | 185 µl | 40- 200 µg | 1 mg | 200 µl |
| 17 cm | 300 µl | 80- 300 µg | 3 mg | 330 µl |
AMERSHAM IPG STRIPS |
| Strip Size | pH 3-10 | pH 4-7 | pH 6-11/6-9 * |
| 11 cm |
20-120 µg
18-1016-61 |
50-300 µg
18-1016-60 |
100-600 µg
17-6001-95 |
| 17 cm |
40-240 µg
17-1235-01 |
75-450 µg
17-1233-01 |
150-900 µg
17-6001-97 |
| * cup load |
|
Loading vol |
Max conc |
| 11 cm |
200 µl |
1 mg |
| 17 cm |
340 µl |
3 mg |
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