Invitrogen™ ZOOM™ Carrier Ampholytes pH 3-10

Catalog No.	Quantity
ZM0021	10 mL

Catalog No. ZM0021 Supplier Invitrogen™ Supplier No. ZM0021

Description

Small, soluble molecules with both positive and negative charge groups

Use Carrier Ampholytes with the ZOOM IPGRunner™ System which consists of three main components: ZOOM IPGRunner Mini-Cell, ZOOM IPGRunner Cassettes and 7cm ZOOM Strips.

• ZOOM Carrier Ampholytes sort based on their isoelectric points in an electric field, thereby establishing a pH gradient in solution or contributing to the gradient when used with IPG strips
• Carrier ampholytes help stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in reproducible IEF resolution

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

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.
• pH Range: pH 3-10
• Quantity: 10 mL
• Shipping Condition: Room Temperature
• Product Line: ZOOM
• Product Type: Carrier Ampholyte

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.

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.

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)

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.

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 should I store the ZOOM Carrier Ampholytes?

We recommend storing the ZOOM Carrier Ampholytes at 4 degrees C. Note: Storing some ZOOM Carrier Ampholytes at 4 degrees C may result in precipitate formation. Dissolve precipitate by warming the solution to 50 degrees C.

What are the storage conditions for the ZOOM Disks, ZOOM Carrier Ampholytes, ZOOM Focusing buffers, ZOOM Cathode buffer, IEF Anode buffer, and IEF Cathode buffer?

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.

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 ampholytes and do you offer them?

Ampholytes are molecules that contain both acidic and basic groups (and are therefore amphoteric) and will exist mostly as zwitterions in a certain range of pH. The pH at which the average charge is zero is known as the molecule's isoelectric point. Ampholytes are used to establish a stable pH gradient for use in isoelectric focusing. In gels containing ampholytes, a linear pH gradient is built up when an electric field is applied. The ampholyte molecules carry a net charge and thus migrate in the electric field between the electrodes as long as they reach the position of corresponding pI. They will then stop moving and form small plateaus (stationary stacks). Commercially available ampholytes are a mixture of synthetic molecules whose individual pI values cover a preselected pH range. Thus one can purchase carrier ampholytes spanning either a wide pH range (e.g., pH 3-10) or a narrow range (e.g., pH 7-8).

We offer ZOOM Carrier Ampholytes that are small, soluble molecules with both positive and negative charge groups. They sort at their relative positions based on their isoelectric points in an electric field, setting up a pH gradient. Carrier ampholytes help stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in reproducible IEF resolution.

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.