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General 2D 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.

We offer the following products for the first- and second-dimension separation of proteins:

First-dimension separation:

Novex™ IEF gels: Vertical gels for separation of proteins based on their isoelectric point (pI)

ZOOM™ IEF Fractionator System: 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

ZOOM™ IPG Runner System: Mini gel system for high-throughput isoelectric focusing of proteins using ZOOM IPG (Immobilized pH Gradient) Strips

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 

The main applications and advantages of performing 2D gel electrophoresis of proteins are listed below:

Applications

  • Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins
  • Protein profiling, biomarker discovery
  • Separation and analysis of protein variants and isoforms

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

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.

ZOOM™ 2D Protein Solubilizers are ready-to-use solutions of proprietary composition (detergents in a chaotrope solution containing urea and thiourea) that enhance solubilization of complex proteins, including hydrophobic and membrane proteins. They are supplied as 1.1X solutions (de-ionized, 0.2 micron filtered,) sufficient for five ZOOM™ IEF Fractionator runs or 100 ZOOM™ IPG Runner runs. We offer ZOOM™ 2D Protein Solubilizer 1 and ZOOM™ 2D Protein Solubilizer 2 that contain different detergent blends and can be used to optimize the sample preparation and determine which solubilizer works best for the sample of choice.

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

IEF Gels

Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins on the basis of their isoelectric point (pI). The pI is the pH at which a protein has no net charge and does not move in an electric field. IEF gels effectively create a pH gradient so proteins separate according to their unique pI.

Novex™ Pre-Cast Vertical IEF gels are excellent for native applications using soluble proteins. They can be used for pI determination and confirmation of isoforms of purified products. They can readily detect minor changes in a protein due to deamination, phosphorylation or glycosylation, and can resolve different proteins of similar size, which cannot be resolved on standard SDS-PAGE gels. Pre-focusing is not required. For a quick 2D analysis, proteins can be separated by pI in the first dimension, using IEF gels, and then by mass in the second dimension, using NuPAGE™ Bis-Tris gels or Novex™ Tris-Glycine gels with a 2D-well or by using ZOOM™ gels for 2D SDS-PAGE.

Novex™ pH 3–10 IEF gels have a pI performance range of 3.5–8.5 and Novex™ pH 3–7 IEF gels have a pI performance range of 3.5–6.5.

Our IEF gels contain 5% acrylamide, 2.6% crosslinker, and 2% ampholytes.

The ratio of acrylamide:bisacrylamide in our IEF gels is 37.5:1.

Our IEF gels do not contain SDS and were not designed for denaturing applications.

The recommended sample loading volumes and protein loading amounts for the different well formats are listed on Page 8 of the Novex™ Pre-Cast Gel Electrophoresis Guide.

We recommend storing them at 4 degrees C.

The IEF buffers should be stored at 4 degrees C.

IEF gels have a shelf life of 8 weeks when stored at 4 degrees C.

During IEF, proteins migrate in an electric field until a stable pH gradient is formed and the proteins settle into their pI. A high finishing voltage is applied to focus the proteins into narrow zones. High voltage cannot be used during the initial stages of IEF as movement of carrier ampholytes generates excessive heat. To obtain the best results, IEF is typically performed by gradually increasing the voltage, then maintaining the final focusing voltage for 30 minutes. So, we recommend: 100 volts for the first hour, 200 volts for the second hour, and 500 volts for the last half hour. Since the voltage will increase over time if the run is set at constant current, we recommend setting the current at 5 mA per gel and setting a voltage limit of 500 volts. Alternatively, IEF can be performed at constant power, so the voltage will increase as the current decreases.

The initial voltage step helps to set up the ampholytes in a pH gradient, the second step actually drives the proteins to their pI and the third step is believed to "fine focus" or sharpen the protein bands.

The use of chilled running buffers will prevent isolated heating and possible gel fissuring at the left and right edges of the gel, thus producing optimal results. The electrophoresis itself can be performed at room temperature.

If the downstream application requires that you don’t use amino acids, you can use 100 mM sodium hydroxide. This will create enough ionic strength for isoelectric focusing.

0.002% methyl red can be added. It runs at pI=3.6, so it is also good for checking how well the gel is running.

We do not recommend adding bromophenol blue to the IEF sample buffer because it is an amphipathic dye that can stick to proteins under native conditions, and cause migration/focusing problems and an increase in current.

Yes, you can reduce the protein with DTT or beta-mercaptoethanol prior to running it on an IEF gel, keeping in mind that if the reducing unfolds or opens masked charges, you may see a change in pI.

The nomenclature of our IEF gels refers to the pI range of ampholytes that are in the IEF gels, and not necessarily the focusing performance range. In practice, a pH 3–10 IEF gel can only resolve proteins with a pI up to about 8.5. This is due to "cathodic drift". During electrophoresis, acrylamide hydrolyzes to polyacrylic acid. This happens to a greater extent at higher pHs than at lower pHs. Consequently, the lower pH acid groups formed at the high pH end of the gradient titrate out the basic groups, lowering the pH gradient at the basic end of the gel to about 8.5. Basic proteins with a pI of 8.5 to 10.0 may migrate into the gel initially, but will migrate backwards out of the gel as the run proceeds.

It is possible run a denaturing IEF gel, but our Novex™ pre-cast vertical IEF gels were not designed for denaturing applications. You can try adding ~7 M urea to the sample prior to running to help solubility. This may work for some proteins but not for others. Further, the protein may re-precipitate since there is no urea in the gel itself, resulting in fuzzy bands and smearing. Use of urea in IEF gels or in running buffers is not generally common because the urea does not remain dispersed throughout the gel. It will migrate to a certain point in the gel and then stop moving.

Yes, this is possible as long as the arginine free base form is used and not the hydrochloric form. Note that the lysine used to make the mixed buffer should be free base lysine; free acid lysine should not be used as a substitute. The reason for adding basic amino acids to the cathode buffer is to increase the ionic strength of the buffer in order to fortify the basic end.

The cathode buffer goes in the inner (upper) buffer chamber and the anode buffer goes in the outer (lower) buffer chamber of the XCell SureLock™ Mini Cell.

The use of chilled running buffers will prevent isolated heating and possible gel fissuring at the left and right edges of the gel, thus producing optimal results. The electrophoresis itself can be performed at room temperature.

No, the SDS will render the protein complex a strong negative charge and therefore an unreliable separation based on isoelectric point. If you have a protein that is moderately soluble, then you can try to solubilize the protein in a non-ionic detergent (between 0.1–0.5%) such as Triton™ X-100, NP-40, or Tween™ 20. The non-ionic detergent may help to solubilize the protein, however, this needs to be tested empirically, because the characteristics of the protein such as its purity, concentration, and ionic strength will influence how well it will run on the gel. Very hydrophobic proteins, like membrane proteins, will not work well, even if a non-ionic detergent is added to the sample buffer.

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.

No. An acetic acid fix is not strong enough. However, a 12% trichloroacetic acid (TCA) fix can be substituted.

For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer.

After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 minutes. Chill the 0.7% acetic acid that will be used as the transfer buffer. Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (–). This is opposite from the typical western blot with SDS, where the negatively charged protein will migrate toward the anode (+) side during the transfer. Transfer for one hour at 10 volts.

Tip for handling the IEF gel: The 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.

ZOOM IEF Fractionator System

The ZOOM™ IEF Fractionator is designed to fractionate samples on the basis of Isoelectric point prior to 2D Electrophoresis, 1D Electrophoresis, or 2D Liquid Chromatography/Mass Spectrometry analysis. The main components of the ZOOM™ IEF Fractionator system are the ZOOM™ IEF Fractionator, ZOOM™ Disks, and ZOOM™ Reagents. Fractionation in the ZOOM™ IEF Fractionator is performed using a series of Sample Chambers connected in tandem and separated by thin membranes (ZOOM™ Disks) containing covalently attached buffers of defined pH. The protein sample is loaded into multiple Sample Chambers separated by these disks or a combination of disks and spacers and subjected to solution-phase IEF for 3 hours. At the end of solution-phase IEF, the protein sample is separated into well-resolved fractions based on the pI of each protein. Samples can be resolved into as many as six fractions (using seven disks of specific pH) from pH 3 to pH 12.

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
  • Novex™ IEF Anode Buffer (50X), 100 mL
  • Novex™ IEF Cathode Buffer pH 3–10 (10X), 2 x 125 mL
  • ZOOM™ Carrier Ampholytes, pH 3–10, 10 mL

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.

ZOOM™ Disks are stable for 3 months when stored at 4 degrees C.

Fractionation helps in reducing sample complexity and enriches low-abundance proteins, thus increasing the dynamic range of detection. We offer the ZOOM™ IEF Fractionator as a simple, fast, and convenient method to reproducibly fractionate cell and tissue lysates into well-resolved fractions on the basis of isoelectric point (pI) using solution-phase isolelectric focusing (IEF). The process can be completed in 3 hours. Fractionated samples are ready for further analysis by two-dimensional gel electrophoresis, one-dimensional gel electrophoresis , or two-dimensional liquid chromatography/mass spectrometry.

ZOOM™ Disks are polyacrylamide gels containing covalently attached buffers of defined pH cast in a fritted polyethylene support. Each ZOOM™ Disk is inserted between Sample Chambers of the ZOOM™ IEF Fractionator to create chambers that will allow separation of proteins in specific pH ranges. For example, inserting ZOOM™ Disks pH 3.0 and pH 4.6 creates a chamber for fractionating proteins between pH 3.0–4.6. 

We offer eight ZOOM™ Disks, ranging from pH 3.0 to pH 12.0. The specific pH for each ZOOM™ Disk is as follows:

pH 3.0, 4.6, 5.4, 6.2, 7.0, 9.1, 10.0, and 12.0.

ZOOM™ Focusing Buffers are proprietary buffer formulations that facilitate optimal fractionation of proteins especially when using the basic ZOOM™ Disks. They include ZOOM™ Focusing Buffer pH 3–7 and ZOOM™ Focusing Buffer pH 7–12. These buffers overcome the background streaking issues associated with the traditional method of using pH 3–10 ampholytes, thus ensuring optimal sample resolution.

Proper sample preparation is the key to the success of IEF fractionation. An ideal sample buffer must maintain the proteins in solution during IEF and not have any effect on the pI of the protein. The sample buffer generally contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT). Due to the large variety of proteins present in different cells and tissues, it is not possible to have a sample preparation protocol that is suitable for all proteins. Based on the starting material and goal of the experiment, the sample preparation protocol needs to be determined empirically. Please refer to the IEF Fractionator Manual for specific sample preparation guidelines.

Our protocol recommends using 99% N,N-Dimethylacrylamide (DMA) for alkylation prior to IEF fractionation. DMA is available from Sigma Aldrich: Cat. No. 274135. We do not recommend using iodoacetamide for alkylation, prior to fractionation, as it will create extremely high currents and poor fractionation results.

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.

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.

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 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 10.0 disks (or pH 12.0 disk for basic proteins) 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.

Yes. They are needed to assist in the migration of the proteins and in the prevention of non-specific protein-protein interactions. One can fractionate without the ampholytes, with the understanding that the protein fractionation patterns may change significantly. 

We would recommend using the ZOOM™ Focusing buffers (pH 3–7 and pH 7–12) for the best results. 

The ZOOM™ IEF Fractionator should be used with an external DC power supply designed for electrophoresis and IEF applications. This power supply must:

  • Be isolated from the ground so that the DC output is floating (safety requirement)
  • Be able to operate at low current (<1 mA) required to perform IEF

Many power supplies automatically shut off when the current drops below 1 mA. You will need a power supply capable of overriding the low current shut-off feature. If the power supply has a current and power limiting capability, we recommend that you set a current limit at 2 mA and a power limit at 2 W.

The electrical leads of the ZOOM™ IEF Fractionator lid are recessed and may not fit into some power supply units. To allow connection of the ZOOM™ IEF Fractionator power leads with certain power supplies, use Novex™ Power Supply Adapters, available separately (Cat. No. ZA10001).

Perform fractionation using the following conditions:
100 V for 20 minutes
200 V for 80 minutes
600 V for 80 minutes

ZOOM IPG Runner System

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.

 

We recommend storing the ZOOM™ Strips at –20 degrees C.

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.

The sample rehydration buffer, also known as the sample buffer, is used to denature and solubilize protein samples, and rehydrate the IPG strips prior to performing IEF using the ZOOM™ IPGRunner system. Due to the large variety of proteins, there is no universal sample rehydration buffer. The sample rehydration buffer must maintain proteins in solution during rehydration of the IPG strips and IEF, and must not have any effect on the pI of the protein. The buffer typically contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT). We offer ZOOM™ 2D Protein Solubilizers that are specifically designed to improve the solubility of hydrophobic and membrane proteins during sample preparation. The ZOOM™ 2D Protein Solubilizer Kit includes two formulations (Solubilizer 1 and 2) that may be used to prepare a sample buffer for a wide variety of proteins. We recommend trying them with your samples.

Note: To obtain high resolution, IEF is usually performed under denaturing and reducing conditions. Use a denaturing sample rehydration buffer for sample preparation (see manual for recipe).

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.

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 5–10 μL. 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.

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.

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.

ZOOM™ Strips pH 9–12 offer a linear pH range of 9–12 that is ideal for isoelectric focusing (IEF) and analysis of basic proteins such as nucleic acid binding proteins, transcription factors, and ribosomal proteins.

We recommend storing them at –80 degrees C. We do not recommend storing them at –20 degrees C.

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.

You can run non-reduced samples in the ZOOM™ IPGRunner by omitting DTT in the rehydration buffer and equilibration buffers.

Our proprietary formulation for our ZOOM™ Strips has been tested and be 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 μLl sample volume. The proteins get in very quickly but the liquid may be left behind. If rehydrating overnight, use the 155 µL sample volume.

The salt concentration should not exceed 10 mM for these reasons:

  • Burning can occur when conductivity is high, hence current limits should be set
  • Protein transport in an electric field decreases with increasing ionic strength.
  • The efficiency of all IEF instruments is decreased when samples contain more than 10 mM salt
  • Longer run times are generally required for samples containing >10 mM salt

Detergents are used in the sample as solubilizing agents:

  • SDS must be 0.2% or lower. If the percentage of SDS is too high, all the proteins will be negatively charged and will migrate to the positive electrode
  • CHAPS can be increased up to 4%
  • NP-40, Triton™-X and other nonionic detergents are used at 0.5–0.7%
  • 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

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™ Novex™ 4–12% Bis-Tris ZOOM™ Gels and Novex™ 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).

The ZOOM™ IPGRunner™ System should be used with an external DC power supply designed for IEF and electrophoresis applications. This power supply must:

  • Be isolated from the ground so that the DC output is floating.
  • Be programmable, with a 4-protocol minimum.
  • Be able to operate at low current (<1 mA) as IEF is performed at very low current.

Note: Many power supplies automatically shut off when the current drops below 1 mA. You will need a power supply capable of overriding the low current shut-off feature.

The electrical leads of the ZOOM™ IPGRunner™ Lid are recessed and may not fit into some power supply units. To allow connection of the ZOOM™ IPGRunner™ power leads with certain power supplies, use Novex™ Power Supply Adapters available separately (Cat. No. ZA10001).