Invitrogen™ ZOOM™ IPGRunner™ Retrofit Kit

Here are the possible causes and solutions:

*Protein has got oxidized: Include DTT in the rehydration buffer and perform the alkylation step.
*Impure solutions: Use ultrapure reagents to prepare the rehydration buffer, equilibration buffer, and buffers for SDS/PAGE.

Here are the possible causes and solutions:

*Impure solutions: Use ultrapure reagents to prepare the rehydration buffer, equilibration buffer, and buffers for SDS/PAGE.
*Air bubble between the strip and 2D gel: Smooth out the air bubbles.
*Poor strip rehydration: Rehydrate the strips in 140 µL rehydrating buffer for 1 hour as described in the manual. Rehydration can be extended to overnight if you use 155 µL rehydrating buffer. Make sure that the rehydration buffer covers the strip completely.
*Protein overload: Decrease the protein concentration or lower the sample volume.
*Protein precipitates: Increase solubilization reagents in the rehydration buffer (see manual). Use appropriate strips based on the pI of the protein sample. Do not add more than 10 µL of your sample to 140 µL of rehydration buffer (see manual).

Here are the possible causes and solutions:

*Improper sample preparation: Increase solubilization reagents in the rehydration buffer. Use 8 M urea for solubilization. Add DTT and non-ionic detergents as described in the manual.
*Incorrect focusing time: Increase or decrease the focusing time based on the initial results.
*High protein load: Decrease the protein load. Use an accurate and sensitive protein estimation method.
*Salt, lipids, or nucleic acid impurities present in the sample: Remove interfering substances (see manual for details).
*Poor strip rehydration: Rehydrate the strips in 140 µL rehydrating buffer for 1 hour as described in the manual. Rehydration can be extended to overnight if you use 155 µL rehydrating buffer. Make sure the rehydration buffer is covering the strip completely.

Here are the possible causes and solutions:

*Low protein load. Increase the protein load. Use an accurate and sensitive protein estimation method.
*Improper sample preparation. Increase solubilization reagents. Use at least 8 M urea for solubilization. Add DTT and non-ionic detergents (see manual for details).
*Strip not correctly oriented. Align the strip correctly as described in the manual. Be sure to have the gel side up when loading the strip into the ZOOM IPGRunner Cassette.
*Air bubbles between the strip and 2D gel. Smooth out any air bubbles.
*Insensitive detection method. Use sensitive detection methods such as silver staining or immunoblotting.

The ZOOM IPGRunner System should be used with an external DC power supply designed for IEF and electrophoresis applications. This power supply must:

*Be isolated from the ground so that the DC output is floating.
*Be programmable, with a 4-protocol minimum.
*Be able to operate at low current (<1 mA) as IEF is performed at very low current.
Note: Many power supplies automatically shut off when the current drops below 1 mA. You will need a power supply capable of overriding the low current shut-off feature.

The electrical leads of the ZOOM IPGRunner Lid are recessed and may not fit into some power supply units. To allow connection of the ZOOM IPGRunner power leads with certain power supplies, use Invitrogen Power Supply Adapters available separately (Cat. No. ZA10001).

Detergents are used in the sample as solubilizing agents:

*SDS must be 0.2% or lower. If the percentage of SDS is too high, all the proteins will be negatively charged and will migrate to the positive electrode.
*CHAPS can be increased up to 4%.
*NP-40, Triton-X and other nonionic detergents are used at 0.5-0.7%.

The salt concentration should not exceed 10 mM for these reasons:

*Burning can occur when conductivity is high, hence current limits should be set.
*Protein transport in an electric field decreases with increasing ionic strength.
*The efficiency of all IEF instruments is decreased when samples contain more than 10 mM salt.
*Longer run times are generally required for samples containing >10 mM salt.

You can run non-reduced samples in the ZOOM IPGRunner by omitting DTT in the rehydration buffer and equilibration buffers.

We do not recommend heating protein samples containing urea over 37 degrees C as elevated temperatures cause urea to hydrolyze to isocyanate, which modifies proteins by carbamylation.

We recommend storing them at -80 degrees C. We do not recommend storing them at -20 degrees C

The recommended ampholyte concentration in the sample rehydration buffer is 0.5%.

*If you are loading 5-50 µg of protein (pure protein or crude lysate) per ZOOM strip, use 0.5% ampholytes in the sample rehydration buffer.
*If you are loading >50 µg of protein (crude lysate or fractionated sample) per ZOOM Strip, use 0.5-2% ampholytes in the sample rehydration buffer.

Note: Higher ampholyte concentration requires longer focusing times.

The maximum volume of the protein sample should at most be 1/6 of the final sample volume that will be added to the strip. A good general volume would be 5-10µL. 140 µL of sample diluted in Sample Rehydration buffer is used to rehydrate each ZOOM Strip for the standard rehydration time of one hour. For overnight rehydration, we recommend using 155 µL of diluted sample.

The sample rehydration buffer, also known as the sample buffer, is used to denature and solubilize protein samples, and rehydrate the IPG strips prior to performing IEF using the ZOOM IPGRunner system. Due to the large variety of proteins, there is no universal sample rehydration buffer. The sample rehydration buffer must maintain proteins in solution during rehydration of the IPG strips and IEF, and must not have any effect on the pI of the protein. The buffer typically contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT).

Note: To obtain high resolution, IEF is usually performed under denaturing and reducing conditions. Use a denaturing sample rehydration buffer for sample preparation (see manual for recipe).

The ZOOM IPGRunner System provides a convenient and quick way to perform isoelectric focusing (IEF) of proteins in a vertical mini-gel format using immobilized pH gradient (IPG) strips for two- dimensional (2D) gel electrophoresis.

The major components of the ZOOM IPGRunner System are:

*ZOOM IPGRunner Mini-Cell (Cat. No. ZM0001)
*ZOOM IPGRunner Cassettes (1 pack of 10 cassettes, Cat. No. ZM0003)
*ZOOM Strips (12 ZOOM Strips, pH 3–10 NL, Cat. No. ZM0011)

Using the ZOOM Strips, proteins are separated based on their isoelectric point (pI). The proteins in the sample can be further separated in the second dimension, based on their molecular weight, by placing the IPG Strips into the IPG well of a NuPAGE Bis-Tris ZOOM gel or Tris-Glycine ZOOM gel. The proteins separated in the second dimension can be visualized as spots by the use of SimplyBlue SafeStain or silver staining, or blotted onto membranes. Protein spots can be also excised from the gel or the membranes to be further analyzed by mass spectrometry or chemical microsequencing to facilitate protein identification.

Alkylation prevents unwanted protein modifications by alkylating cysteines to avoid mixed disulfide formation and reoxidation and this allows for crisper focusing.

Sorry we have discontinued selling the ZOOM 2D Protein Solubilizers.

The main advantages of performing 2D gel electrophoresis of proteins and applications used for are listed below:

Advantages:

*Simultaneous separation of hundreds to thousands of proteins
*High capacity with superior resolution
*Compatible with further analysis by MS for protein identification and sequencing
Ability to separate and analyze low-abundance proteins

Applications:

*Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins
*Protein profiling, biomarker discovery
*Separation and analysis of protein variants and isoforms

We offer the following products for the first- and second-dimension separation of proteins:

First-dimension separation:

*Vertical gels for separation of proteins based on their isoelectric point (pI) (https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/protein-gels/specialized-protein-separation/isoelectric-focusing.html)

*Solution-phase isoelectric focusing of proteins using ZOOM Disks (immobilized buffer disks of specific pH) to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/isoelectric-focusing/zoom-ief-fractionator.html)

*Mini gel system for high-throughput isoelectric focusing of proteins using ZOOM IPG (Immobilized pH Gradient) Strips (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/2d-gel-electrophoresis/zoom-ipgrunner-system.html)

Second-dimension separation:

*ZOOM gels for 2D electrophoresis: NuPAGE Bis-Tris (Cat. No. NP0330BOX) and Tris-Glycine (Cat. No. EC60261BOX) mini gels with IPG wells ( to accommodate 7 cm ZOOM strips) for separation of proteins based on their molecular weight

2D protein gel electrophoresis is the separation of proteins in two dimensions. In the first dimension, proteins are separated by their isoelectric point (pI) using isoelectric focusing, and in the second dimension, they are separated by their mass using SDS-PAGE.