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View additional product information for ZOOM™ IPGRunner™ Cassette - FAQs (ZM0003)
25 product FAQs found
一个可能的原因是电极间连接不良或电路未闭合。应确保在Electrode Wicks中加入600 μL去离子水，并且凝胶暴露于凝胶盒的阳极和阴极端。检查电源。确保关闭电源的“负载检测”功能，使电源能够在低电流下运行。
以下是ZOOM IPG Runner Mini-Cell的组件：
•ZOOM IPGRunner 核心
•ZOOM IPGRunner盖子
•凝胶张力楔
•缓冲液挡板
•Mini-Cell槽
There are several reasons why streaking may occur.
(1) Sample is not completely solubilized prior to application.
(2) Sample is poorly soluble in rehydration solution.
(3) Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2-D result, particularly toward the acidic side of the gel.
(4) Ionic impurities are present in sample.
(5) Ionic detergent is present in sample.
(6) Sample load is too high.
(7) Underfocusing. Focusing time was not long enough to achieve steady state focusing.
(8) Overfocusing. Extended focusing times (over 100,000 Vh) may result in electroendosmotic water and protein movement, which can produce horizontal smearing.
What should be done?
(1) Be sure that the sample is completely and stably solubilized. Note: Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.
(2) Increase the concentration of the solubilizing components in the rehydration solution.
(3) Modify sample preparation to limit these contaminants or dialyze protein.
(4) Reduce salt concentration to below 10 mM by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides and other small molecules.
Note: Specific and non-specific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples. If the sample preparation cannot be modified, the effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100-150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip.
(5) If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the non-ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins.
(6) Extend focusing time. Load less sample.
(7) Prolong focusing time.
(8) Reduce focusing time.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
A possible reason is poor contact between electrodes or incomplete circuit. Make sure that you have added 600 µL deionized water to the Electrode Wicks and the gel is exposed at the anodic and cathodic ends of the cassette. Check the power supply. Be sure to set the “Load Check” to off to enable the power supply to operate at low current.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are the components of the ZOOM IPGRunner Mini-Cell:
ZOOM IPGRunner Core
ZOOM IPGRunner Lid
Gel Tension Wedge
Buffer Dam
Mini-Cell Chamber
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are the possible causes and solutions:
*Protein degradation: Add protease inhibitors during sample preparation (see manual for details).
*Different subunits: Use denaturing conditions (8 M urea).
*Incomplete equilibration: Perform equilibration as described in the manual. Increase the equilibration time.
*Protein precipitates: Increase solubilization reagents in the rehydration buffer (see manual for details. Use appropriate strips based on the pI of the protein sample.
*Low protein load: Increase the protein load. You can load up to 400 µg of fractionated protein sample per ZOOM Strip. Use an accurate and sensitive protein estimation method.
*Insensitive detection method: Use sensitive detection methods such as silver staining or immunoblotting.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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%.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
You can run non-reduced samples in the ZOOM IPGRunner by omitting DTT in the rehydration buffer and equilibration buffers.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend storing them at -80 degrees C. We do not recommend storing them at -20 degrees C
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Alkylation prevents unwanted protein modifications by alkylating cysteines to avoid mixed disulfide formation and reoxidation and this allows for crisper focusing.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Sorry we have discontinued selling the ZOOM 2D Protein Solubilizers.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.