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Invitrogen™ ZOOM™ IPG Strips, pH 3-10L

Catalog No. ZM0018
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ZM0018 12 strips
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Catalog No. ZM0018 Supplier Invitrogen™ Supplier No. ZM0018
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Description

Requires

ZOOM IPGRunner System

Oil-free, trouble-free 2D electrophoresis in a mini-gel format

  • First dimension separation, isoelectric focusing (IEF), is complete in less than three hours
  • The mini-gel design of the system is easy to handle, eliminates mineral oil overlays, and allows processing of up to 12 samples at once for high-throughput usage
  • Strips are 7cm long and contain a thin layer of polyacrylamide gel that includes a fixed pH gradient
  • Each strip is clearly labeled with a unique identifying number, pH range, and orientation marks
  • Strips are supplied attached to a tri-fold card for easy access and removal

2D Gel Electrophoresis, IsoElectric Focusing, Protein Gel Electrophoresis, Protein Sample Fractionation, Protein Sample Preparation and Protein Purification, Proteins, Expression, Isolation and Analysis

Order Info

Shipping Condition: Dry Ice

Specifications

Content And Storage The ZOOM™ IPGRunner™ Mini-Cell includes the ZOOM™ IPGRunner™ Buffer Core and Lid, Gel Tension Wedge, Buffer Dam, and the Mini- Cell chamber. The ZOOM™ IPGRunner™ Combo Kit includes the ZOOM™ IPGRunner™ Mini-Cell, electrode wicks, sealing tape, 10 ZOOM™ IPGRunner™ Cassettes, and 12 ZOOM™ Strips, pH 3-10 NL. The ZOOM™ IPGRunner™ Retrofit Kit for XCell SureLock™ Mini-Cell includes the ZOOM™ IPGRunner™ Buffer Core and Lid. The ZOOM™ IPGRunner™ Cassettes include electrode wicks and sealing tape.
Gel Size Mini
pH Range 3.0 - 10.0 (linear)
Quantity 12 strips
Shipping Condition Dry Ice
Product Line ZOOM
Product Type IPG Strip
For Use With (Equipment) XCell SureLock Mini-Cell

Frequently Asked Questions (FAQs)

What may cause streaking on the 2nd dimension gel after IEF?

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.
What is the protocol for using ZOOM gels after first-dimension separation on an IPG strip?

1) Trim excess plastic from IPG strip

2) Equilibrate IPG strip for 2 x 15 min in 5 mL of the appropriate buffer (Use NuPAGE sample buffer for NuPAGE ZOOM gels and Tris Glycine ZOOM gels). Equilibration buffer: 1X NuPAGE sample buffer, 50 mM DTT.

3) Place equilibrated IPG strip in large well of ZOOM gel. Seal the strip in place with an agarose (0.5%) overlay.

4) Load molecular weight markers in small well of ZOOM gel.

Variations: To alkylate the proteins after reducing them, prior to separation on ZOOM gels: 1) Incubate IPG strip for 15 min in equilibration buffer 2) Transfer strip to Equilibration Buffer containing 125 mM iodoacetamide and lacking reducing agent. Incubate an additional 15 min. To denature the proteins with urea after IEF: 6 M urea can be added to Equilibration Buffer, if desired.
How should I equilibrate ZOOM strips before performing SDS-PAGE using ZOOM gels?

Incubating ZOOM Strips in NuPAGE LDS Sample Buffer equilibrates the strips in SDS buffer and prepares the strips for 2D SDS-PAGE.

We recommend using the NuPAGE LDS Sample Buffer containing 50 mM DTT (NuPAGE Sample Reducing Agent) with NuPAGE Invitrogen 4-12% Bis-Tris ZOOM Gels and Invitrogen 4-20% Tris-Glycine ZOOM Gels. You need 5-15 mL of buffer per equilibration tray. For alkylation, we recommend incubating the strips in 125 mM Alkylating Solution (prepared by dissolving 232 mg of fresh iodoacetamide in 10 mL of 1X NuPAGE LDS Sample Buffer).

What is your recommendation for the extraction buffer for my protein sample after IEF using ZOOM IPG strips?

*The rehydration buffer is the best extraction buffer for IPG strips.
*The ionic strength of the buffer should be low; e.g., PBS will not work because the ionic strength is pretty high (150-250 mM), depending on salt and other ionic components.
*Proteins can be precipitated and re-suspended in rehydration buffer; use acetone for precipitation.

Can I rehydrate my ZOOM IPG strip for less than 8-16 hours?

Our proprietary formulation for our ZOOM Strips has been tested and been shown to allow for a 60-90 minute rapid rehydration step. It is not necessary to go overnight, although for convenience, you still can rehydrate for 8-16 hours. For the one hour rehydration, it is important not to exceed the 140 µL sample volume. The proteins get in very quickly but the liquid may be left behind. If rehydrating overnight, use the 155 µL sample volume.

Can I heat my protein sample prepared using ZOOM 2D Protein Solubilizer?

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.

How do you recommend storing protein samples prepared in the Sample Rehydration buffer?

We recommend storing them at -80 degrees C. We do not recommend storing them at -20 degrees C
How much ampholyte do you recommend adding to the sample rehydration buffer?

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.

How can I store the ZOOM IPG strips after running the first dimension?

You can store the whole cassette at -80 degrees C in a sealed container until ready to use. However, it is possible to put the individual strips in 15 mL conical tubes and freeze them at -80 degrees C. When you are ready to equilibrate the strip before running the second dimension, you can add the equilibration buffer directly to the tube. In addition, you can remove the film cover from the cassette and store the strips, in the cassette, in a zip-sealed bag for ease of removal of single strips. Do not allow the other strips to thaw and then refreeze because it might result in diffusion of the bands. The strips can be removed frozen.

What is the maximum recommended volume of protein sample to be added to the Sample Rehydration buffer?

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.

How much protein do you recommend loading on a ZOOM Strip?

We recommend starting with 5-15 µg (for silver staining) or 20-50 µg (for Coomassie staining) of total protein per ZOOM Strip. The total protein load can be increased after optimizing the sample preparation protocol and focusing parameters. Higher amounts of sample may be loaded on narrow pH range ZOOM Strips. For the ZOOM Strip pH 9-12, 50-100 µg (for silver staining) or 100-200 µg for (Coomassie staining) of total protein per strip is recommended. If the sample is a fractionated or partially purified protein, up to 400 µg of total protein per strip may be loaded.

How should I store the ZOOM Strips?

We recommend storing the ZOOM Strips at -20 degrees C.

What is the purpose of alkylation prior to IEF?

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

Do you still offer ZOOM 2D Protein Solubilizers?

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

What are the main reasons for performing 2D gel electrophoresis of proteins?

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

What products do you offer for 2D gel electrophoresis of proteins?

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

What is 2D protein gel electrophoresis?

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.

For Research Use Only. Not for use in diagnostic procedures.