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Store the fixed, stained, and destained gel strip in 8% acetic acid at 4-25 degrees C in a sealed dish. Do not freeze as this will cause the gel strip to crack.

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The length of the 2D well is 6.5 cm. The IEF gel strip must be trimmed down to 5.8-5.9 cm before positioning it into the 2D well, even after it has been soaked in methanol to shrink the gel. For the 7 cm IPG strips, significant trimming would be necessary if the gels with the 2D wells are to be used. For IPG strips, the longer IPG well (found in the ZOOM gels) is recommended.

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The PowerEase Touch HV Power Supply is ideal for high voltage separation such as isoelectric focusing (IEF gels and IPG Strips) and 2-D electrophoresis. In addition, it also supports DNA/RNA electrophoresis.

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SYPRO Ruby Protein Gel Stain binds primarily to proteins through ionic charges of the dye, with basic side chains (lysine, arginine, histidine and to a lesser extent with tyrosine and tryptophan. The fixative solution and SYPRO Ruby stain solution both have an acidic pH, and SYPRO Ruby dye binding increases with protonated basic residues. SYPRO Ruby dye will also bind SDS bound to the proteins and in the gel matrix. The high 50% methanol concentration in the fixative solution is better at stripping out the SDS from the gel matrix, lowering the background staining and allowing for an optimal signal to noise.

Here is a reference on amino acid specificity of SYPRO Ruby Protein Gel Stain:
Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Steinberg TH, Chernokalskaya E, Berggren K, Lopez MF, Diwu Z, Haugland RP, Patton WF. Electrophoresis 2006, 21, 486-496.

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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.

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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.

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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

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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.

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The ZOOM IPGRunner System provides a convenient and quick way to perform isoelectric focusing (IEF) of proteins in a vertical mini-gel format using immobilized pH gradient (IPG) strips for two- dimensional (2D) gel electrophoresis.

The major components of the ZOOM IPGRunner System are:

*ZOOM IPGRunner Mini-Cell (Cat. No. ZM0001)
*ZOOM IPGRunner Cassettes (1 pack of 10 cassettes, Cat. No. ZM0003)
*ZOOM Strips (12 ZOOM Strips, pH 3–10 NL, Cat. No. ZM0011)

Using the ZOOM Strips, proteins are separated based on their isoelectric point (pI). The proteins in the sample can be further separated in the second dimension, based on their molecular weight, by placing the IPG Strips into the IPG well of a NuPAGE Bis-Tris ZOOM gel or Tris-Glycine ZOOM gel. The proteins separated in the second dimension can be visualized as spots by the use of SimplyBlue SafeStain or silver staining, or blotted onto membranes. Protein spots can be also excised from the gel or the membranes to be further analyzed by mass spectrometry or chemical microsequencing to facilitate protein identification.

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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.

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