ZOOM™ IEF Fractionator Combo Kit - FAQs

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。

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。

检查样品槽或凹槽是否有任何损坏。使用备件盒中的另一个样品槽。

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。

阳极端密封器连接有一个黑色的环状摩擦密封圈。如果难以将阳极端密封器插入槽组装管中，可去掉黑色的环状摩擦密封圈。

以下是可能原因和解决方案：

- 缓冲液槽内使用了错误的缓冲液。根据使用手册的说明，使用稀释的阳极和阴极缓冲液。我们推荐使用能够设置功率和电流极限的电源，以避免过高的电流对组分分离器造成意外损害。
- 高盐浓度。将样品中的盐浓度限制在10mM或更低。

以下是可能原因和解决方案：

- 缓冲液槽内使用了错误的缓冲液。根据使用手册的说明，使用稀释的阳极和阴极缓冲液。如果您自己配制阳极和阴极缓冲液（见使用手册中的配方），应使用赖氨酸（游离碱）和精氨酸（游离碱）。不要使用赖氨酸HCl和精氨酸HCl。
- 试剂质量差或尿素降解。制备样品和缓冲液时，应使用高质量、蛋白质组学级别的试剂。应使用新配制的或在-80℃冻存的尿素溶液。按照生产商的说明，在混合离子交换树脂上将尿素溶液去离子化。

以下是可能原因和解决方案：

- 低电流关闭功能开启。检查电源。按照生产商的说明，禁用低电流关闭功能，从而使电源在低电流下也能运行。
- 槽内存在气泡。避免槽组装管或样品槽内存在气泡。如果有气泡，使用凝胶上样滴管将其刺破。

我们推荐每次使用完毕后先用温和的洗涤剂清洗ZOOM IEF组分分离器，再用去离子水冲洗。盖子上的电线插头插入电源时，不要清洗盖子。

ZOOM IEF组分分离器不受酒精的影响，但不能兼容氯化烃（如氯仿）、芳香烃（如甲苯和苯）或丙酮。

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。

No. That much formic acid will overpower the sample buffer, the pH will drop, and the SDS will precipitate. A back-titration with Tris base or sodium hydroxide may help, but a lot depends on the volume of sample to be loaded. A load of 2-3 µL may possibly work.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Since this is an end fraction, and you are only interested in the sample in one chamber, it is okay to leave this chamber without sample (include buffer, however). This will help ensure that only the basic proteins will migrate into this chamber and will give cleaner results, e.g., this will avoid the problem of having the very acidic proteins clogging up the 9.1 disk, as they would be exiting into the 9.1-3.0 chamber and possibly interacting with the very basic proteins entering the 9.1-12.0 chamber. Lysate and buffer are needed in the rest of the fractionator.

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Nitrocellulose filters can be used, but they do contain certain charged chemicals that may seep into the buffer and may contribute to high conductivity. PVDF does not have this issue.

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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.

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This is not recommended. Excess heat may be generated.

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We recommend loading 650 µL sample per chamber. Lower volumes do not produce consistent results. This is potentially due to lateral diffusion of sample proteins into the portion of the disk that is not exposed to the electric field, which leads to less sharp cutoffs.

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Proteins are ampholytes, but in general they are poor carrier ampholytes. The profiles of the fractions are similar, but not identical, to those obtained with ampholytes in the sample.

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We do not recommend using NaOH. It is too harsh for the pH 10 disks, it decomposes, and it allows for more carbonates to build up in the system during the run.

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Not necessarily; there are several options:

(1) Use the same volume of sample in fewer chambers [compensating spacers for disks]

(2) Use a higher volume of sample by using even more spacers [equivalent to increasing from 5 chambers to 6 or 7]

(3) Use less sample volume and more of the anode and cathode buffers

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Yes, we recommend 0.6 mg/mL, however good separation has been obtained with 650 µL of 2.4 mg/mL rat liver lysate in IEF denaturant.

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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.

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No, all the chambers need to be filled with buffer. Otherwise, there would be a break in the current flow within the apparatus.

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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 you 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 disks 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.

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During the run at the parameters suggested, the ZOOM IEF Fractionator will never produce more than 1 Watt. Using the limits suggested in the manual, this unit will not generate heat to more than a couple of degrees. There is not sufficient heat generated to result in protein modifications such as carbamylation. Running it for overnight, however, may result in protein carbamylation.

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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.

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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.

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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.

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Inspect the Sample Chamber to check any damage to the Sample Chamber or groove. Use another Sample Chamber included in the Spares Box.

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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.

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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.

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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.

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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, proteomic-grade reagents for sample and buffer preparation. Use freshly prepared urea solutions or stored frozen at -80 degrees C. Deionize urea solutions on a mixed bed ion exchanger resin using manufacturer's recommendations.

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Here are 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.

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We recommend washing the ZOOM IEF Fractionator with a mild detergent and rinsing with deionized water after each use. Do not wash lid with cables plugged into the power supply.

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The ZOOM IEF Fractionator is impervious to alcohol, but not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene) or acetone.

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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.

The ZOOM IEF Fractionator Combo kit includes the following components:

*ZOOM IEF Fractionator
*ZOOM Disks pH 3.1, 4.6, 5.4, 6.2, 7.0, 10.0 (one pack of 10, each)
*ZOOM Urea, 1 kg
*ZOOM Thiourea, 0.5 kg
*ZOOM CHAPS, 5 g
*Invitrogen IEF Anode Buffer (50X), 100 mL
*Invitrogen IEF Cathode Buffer pH 3-10 (10X), 2 x 125 mL
*ZOOM Carrier Ampholytes, pH 3-10, 10 mL

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.