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查看更多产品信息 ZOOM™ IEF Disk pH 3.0 - FAQs (ZD10030)
40 个常见问题解答
这可能是因为样品槽间发生泄漏。请根据使用手册中的说明，将样品槽安装到槽组装管中。分离器组装不恰当，会导致密封不良，引起组分泄漏和污染。应按照使用手册的说明，将样品槽环状密封器正确插入到样品槽的凹槽中，并将ZOOM Disk放到槽上。
尝试用棉签在密封圈周围轻轻涂抹硅胶，润滑阴极槽密封器（黑色环状）。大部分实验通常都有硅胶。如果槽密封器损坏，则使用备件盒中新的槽密封器更换。
去掉环状摩擦密封圈可能导致阳极端密封器和样品槽滑入槽组装管中。如果发生这种情况，则在阳极端密封器上添加一个环状摩擦密封圈。
我们提供以下蛋白质凝胶染色盘：
•StainEase 染色盘（货号NI2400）可用于对小型和中型凝胶进行方便、均匀的染色。该染色盘组件包括一个用于放置凝胶和排出染料的多孔滤管嵌件、一个盖子以及不必操作凝胶即可在其中更换染料和溶液的外部容器。可轻松安装最多4块小型凝胶。
•小型凝胶孵育盘（货号22843）可用于小型凝胶和蛋白免疫印迹膜的染色。
Between 65% and 90% of the protein loaded can be recovered without using a washing step. With a washing step (detailed in the manual for the ZOOM IEF Fractionator), 95% of the protein can be recovered.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Upon receipt, store ZOOM Disks at 4 degrees C. We guarantee a minimum of 3 months of usable shelf life for the ZOOM discs when they are stored properly.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This could potentially be due to leakage between Sample Chambers. Assemble the Sample Chambers in the Chamber Assembly Tube as described in the manual. Improper assembly of the fractionator will not produce proper sealing and result in leaking and contamination of fractions. Be sure to properly insert the Sample Chamber O-ring Seals on the groove of the Sample Chamber and place the ZOOM Disks on the chamber as shown in the manual.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Try lubricating the Cathode Chamber Seal (black O-ring) with silicone by lightly dabbing silicone around the seal with a swab. Silicone is typically available in most laboratories. If the Chamber Seal is damaged, replace with a new Chamber Seal included in the Spares Box.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Inspect the Sample Chamber to check any damage to the Sample Chamber or groove. Use another Sample Chamber included in the Spares Box.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
A black friction O-ring is attached to the Anode End Sealer. If the Anode End Sealer is difficult to insert into the Chamber Assembly Tube, remove the black friction O-ring.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The ZOOM IEF Fractionator includes the following components:
Chamber Assembly Tube with Anode Reservoir
Spill Trough with Cathode Reservoir Lid
Sample Chambers (7)
Sample Chamber O-ring Seals, red (10)
Sample Chamber Caps with O-rings (7)
Cathode End Sealer
Anode End Sealer
Cathode End Screw Cap
Spacers, black (8)
Spares Box 1
Sample Chamber O-ring Seals (8)
Sample Chamber Caps with O-rings (7)
Spares Box 2
Cathode Chamber Seals (2)
Spacers, black (8)
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
This is normal and is due to the negatively charged phospholipids in a sample that move toward the anode, that are not very soluble at the low pH. If cloudiness is seen at the cathode end, presumably it is due to positively charged lipids that aren't soluble at the high pH of the cathode chamber.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
This is likely due to the proteins not being alkylated properly or that they are at the verge of being insoluble. If proteins are not alkylated properly, they may associate with any number of proteins and be found in every fraction at the end of the run. If they are on the verge of being insoluble, it may be that they are sticking to the gel in which case, stronger chaotropes may be needed.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
If the alkylation step is left out after reduction with DTT, streaking in the final results may be observed. The failure to alkylate may result in spots in the alkaline pH region due to scrambled disulfide bridges among like and unlike chains. As reduced polypeptide chains migrate toward their pI, they leave behind the reducing agent, and they may be re-oxidized and re-form disulfide bridges. The regenerated disulfide bridges may occur between unlike polypeptide chains, resulting in spots that do not represent the true protein profile.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Our protocol recommends using 99% N,N-dimethylacrylamide (DMA) for alkylation prior to IEF fractionation. DMA is available from Sigma Aldrich, Cat. No. 274135. We do not recommend using iodoacetamide for alkylation, prior to fractionation, as it will create extremely high currents and poor fractionation results.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Removal of the friction O-ring may result in sliding of the Anode End sealer and Sample Chambers into the Chamber Assembly Tube. If this results, add the friction O-ring on the Anode End Sealer
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are the possible causes and solutions:
*Incorrect buffers used in the buffer reservoirs. Use diluted anode and cathode buffers as described in the manual. We recommend using a power supply capable of setting power and current limit to avoid accidental damage to the fractionator due to high currents.
*High salt concentration. Limit the salt concentration in the samples to 10 mM or less.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are the possible causes and solutions:
*Incorrect buffers used in the buffer reservoirs. Use diluted anode and cathode buffers as described in the manual. If you are preparing your own anode and cathode buffers (see manual for a recipe), use lysine (free base) and arginine (free base). Do not use lysine HCl and arginine HCl.
*Poor quality reagents used or urea is degraded. Use high-quality, proteomics-grade reagents for sample and buffer preparation. Use freshly prepared urea solutions or stored frozen at -80 degrees C. De-ionize urea solutions on a mixed bed ion exchanger resin using manufacturer's recommendations.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are the possible causes and solutions:
*Low current shut-off feature enabled. Check the power supply. Be sure to override the low current shut-off feature as recommended by the manufacturer to enable the power supply to operate at low current.
*Air-bubble in chambers. Avoid trapping any bubbles in the Chamber Assembly Tube or in Sample Chambers. If there are any bubbles, use a gel loading tip to break the bubbles.
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.
We would recommend using the ZOOM Focusing buffers (pH 3-7 and pH 7-12) for the best results.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes. They are needed to assist in the migration of the proteins and in the prevention of non-specific protein-protein interactions. One can fractionate without the ampholytes, with the understanding that the protein fractionation patterns may change significantly.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, you can use less than the full complement of ZOOM Disks if you're only interested in fractionating over a narrow pH range and want to concentrate your fraction of interest. Use a Spacer instead of a ZOOM Disk between the chambers. It is recommended that you still use the pH 3.0 and the pH 10.0 disks (or pH 12.0 disk for basic proteins) at the anode and cathode ends to create a pH gradient for efficiently removing the high- and low-pI proteins away from the fraction of interest.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
It will migrate to the most acidic chamber (pH 3.0-4.6), unless its pI is less than 3.0, in which case, it will migrate into the anode tank.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Fractionated samples can be further separated in the first dimension, on a series of overlapping narrow pH range ZOOM Strips. The fractionated sample is in the same buffer required for IEF using ZOOM Strips. We recommend using narrow pH range ZOOM Strips, which are ~0.1 pH unit wider than the fractionated pools. For example, first dimension IEF of fraction pH 4.6-5.4 from the fractionator is performed using ZOOM Strip pH 4.5-5.5. Each fraction can provide enough focused sample to load four 7-cm ZOOM IPG Strips. Alternatively, the fractionated sample can be run on an IEF gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Our protocol recommends using 99% N,N-Dimethylacrylamide (DMA) for alkylation prior to IEF fractionation. DMA is available from Sigma Aldrich: Cat. No. 274135. We do not recommend using iodoacetamide for alkylation, prior to fractionation, as it will create extremely high currents and poor fractionation results.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Proper sample preparation is the key to the success of IEF fractionation. An ideal sample buffer must maintain the proteins in solution during IEF and not have any effect on the pI of the protein. The sample buffer generally contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT). Due to the large variety of proteins present in different cells and tissues, it is not possible to have a sample preparation protocol that is suitable for all proteins. Based on the starting material and goal of the experiment, the sample preparation protocol needs to be determined empirically. Please refer to the manual (http://tools.thermofisher.com/content/sfs/manuals/zoomieffractionator_man.pdf) for specific sample preparation guidelines.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
ZOOM Disks are polyacrylamide gels containing covalently attached buffers of defined pH cast in a fritted polyethylene support. Each ZOOM Disk is inserted between Sample Chambers of the ZOOM IEF Fractionator to create chambers that will allow separation of proteins in specific pH ranges. For example, inserting ZOOM Disks pH 3.0 and pH 4.6 creates a chamber for fractionating proteins between pH 3.0-4.6.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Fractionation helps in reducing sample complexity and enriches low-abundance proteins, thus increasing the dynamic range of detection. We offer the ZOOM IEF Fractionator as a simple, fast, and convenient method to reproducibly fractionate cell and tissue lysates into well-resolved fractions on the basis of isoelectric point (pI) using solution-phase isolelectric focusing (IEF). The process can be completed in 3 hours. Fractionated samples are ready for further analysis by two-dimensional gel electrophoresis, one-dimensional gel electrophoresis , or two-dimensional liquid chromatography/mass spectrometry.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
ZOOM Disks are stable for 3 months when stored at 4 degrees
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend storing the ZOOM Disks, ZOOM Carrier Ampholytes, ZOOM Focusing buffers, ZOOM Cathode buffer, IEF Anode buffer, and IEF Cathode buffer at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The ZOOM IEF Fractionator is designed to fractionate samples on the basis of Isoelectric point prior to 2D Electrophoresis, 1D Electrophoresis, or 2D Liquid Chromatography/Mass Spectrometry analysis.
The main components of the ZOOM IEF Fractionator system are the ZOOM IEF Fractionator, ZOOM Disks, and ZOOM Reagents. Fractionation in the ZOOM IEF Fractionator is performed using a series of Sample Chambers connected in tandem and separated by thin membranes (ZOOM Disks) containing covalently attached buffers of defined pH. The protein sample is loaded into multiple Sample Chambers separated by these disks or a combination of disks and spacers and subjected to solution-phase IEF for 3 hours. At the end of solution-phase IEF, the protein sample is separated into well-resolved fractions based on the pI of each protein. Samples can be resolved into as many as six fractions (using seven disks of specific pH) from pH 3 to pH 12.
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.