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ZOOM™ IEF Fractionator Combo Kit - FAQs

ZOOM™ IEF Fractionator Combo Kit - FAQs

View additional product information for ZOOM™ IEF Fractionator Combo Kit - FAQs (ZF10001)

27 product FAQs found

The final concentration of formic acid in the sample that I am loading on a Bis-Tris gel will be 5%. Will this work?

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.

I will be using the 3-disk extended format for the assembly of the fractionator. When I load my samples, the manual tells me not to add my sample buffer with lysate to the pH 9-12 chamber. I am trying to isolate a protein at pH 10, so wouldn't it make more sense to add my sample to this chamber?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

In the appendix section of ZOOM IEF Fractionator manual that details making the 1.1X IEF Denaturant, it states to filter the supernatant using a 0.2 um filter and to use polyvinylidene fluoride (PVDF) or nylon filters but not nitrocellulose or cellulose acetate filters. Why can't nitrocellulose be used?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What amount of protein can be recovered from the ZOOM IEF Fractionator?

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.

When using the ZOOM IEF Fractionator, is there any downside to increasing the voltage at the beginning of the run as long as the 2 mA current limit is not exceeded?

This is not recommended. Excess heat may be generated.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I load less than the recommended 650 µL sample volume into the chambers of the ZOOM IEF Fractionator?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

When running the ZOOM IEF Fractionator, if there is not a solubility issue, will the proteins/peptides in the sample migrate to their expected chambers based on isoelectric point in the absence of carrier ampholytes? Won't they act as ampholytes within each chamber?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can you replace the lys/arg cathode buffer with an equivalent concentration of sodium hydroxide in the ZOOM IEF Fractionator?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

If you leave out chambers in the ZOOM IEF Fractionator, is it necessary to compensate by increasing the volume of the anode and cathode buffers?

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can more than the recommended 0.6 mg/mL protein sample be loaded into the ZOOM IEF Fractionator?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What happens if the protein sample to be loaded into the ZOOM IEF Fractionator is acidic?

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 Assays and Analysis Support Center.

Can the ZOOM IEF Fractionator be run with one or more of the chambers empty?

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I use less than the full set of ZOOM Disks with the ZOOM IEF Fractionator? I am only interested in the 4.6-5.4 fraction.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How much heat is generated when running samples on the ZOOM IEF Fractionator? Will it affect my proteins?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I just set up the ZOOM IPGRunner for IEF but don't see any current running through the system. What is the issue?

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.

I performed IEF fractionation using the ZOOM IEF Fractionator and the fractions are not well-defined. What is the problem?

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.

While assembling the ZOOM IEF fractionator, the Chamber Assembly Tube is difficult to insert or remove from the cathode reservoir. Do you have any suggestions?

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.

While assembling the ZOOM IEF fractionator, the Sample Chamber O-ring Seal does not fit into the Sample Chamber. Can you offer some tips?

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.

The Anode End Sealer seems to be sliding into the Chamber Assembly Tube of the ZOOM IEF fractionator during assembly. What can I do?

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.

I am finding it difficult to insert the Anode End Sealer into the Chamber Assembly Tube of the ZOOM IEF fractionator? Can you offer some tips?

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.

I started my IEF fractionation using the ZOOM IEF Fractionator and the starting current is very high. What could cause this?

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.

When I started my IEF fractionation using the ZOOM IEF Fractionator, the current was low but I just noticed that it is very high now. What could have caused this?

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.

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

I started my IEF fractionation using the ZOOM IEF Fractionator and just noticed that there is no current passing through the system. Can you please help me troubleshoot?

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.

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

What do you recommend for regular maintenance of the ZOOM IEF Fractionator?

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.

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

What is the tolerance of the ZOOM IEF Fractionator to organic solvents?

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.

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

What are the components of the ZOOM IEF Fractionator?

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.

What are the components of the ZOOM IEF Fractionator Combo kit?

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.