In the last two posts- 2-Dimensional PAGE and Sample Preparation In 2-D PAGE, I have written about the 2-D PAGE. In the first post on 2-D PAGE, I had given a brief summary and in the second one, I had elaborated on the sample preparation procedure. In this post I am going to elaborate on the IPG strip Rehydration.
IPG strip rehydration
Immobilized pH gradient (IPG) strips are dehydrated and must be rehydrated to their original gel thickness (0.5 mm) before use.
Several recipes of sample buffer or rehydration buffer available. The choice of the rehydration buffer depends on experimental condition and targeted proteins of interest. However a typical solution generally contains urea, non-ionic or zwitterionic detergent, dithiothreitol (DTT), Pharmalytes appropriate to the pH range of the IPG strip and dye (each ingredient explained later).
Purified proteins are added to the rehydration buffer. Addition to the rehydration buffer helps in protein solubilization and loading into the gel for separation. The volume of rehydration buffer required can be calculated by the number of the strips used for the run accordingly.
The Rehydration can be of two types based on the use of electric field: Passive and Active Rehydration. During rehydration the cover fluid (a mineral oil) has to be added to the strip (without air bubbles), to prevents evaporation and attain the set voltage across all the wells.
- Passive Rehydration
This is an overnight process, that helps the IPG strip to get rehydrated with protein sample solution inabsence of electric field. In this, proteins enter the gel by absorption only. This method allows efficient use of equipment since strips can be rehydrated in sample rehydration trays while other samples are being focused in the IEF cell. Once the passive rehydration is over, the strip is ready for IEF run.
In active rehydration, the rehydration is carried out in the presence of electric field. A very low voltage is applied during rehydration of the strips. Proteins enter the gel matrix under current as well as by absorption. Active rehydration is thought to help large proteins enter the strip by applying electrical “pull“. Because the voltage is applied before all the solution and proteins are absorbed into the gel, the pH of a protein’s environment will be the pH of the rehydration buffer, and the protein will move according to its mass-to-charge ratio in that environment. Thus, small proteins with a higher mobility have a higher risk of being lost from the strip. Sample is loaded in a single manifold electrode unit in case of single strip run or strips are loaded in the 12-well manifold. In active process, the condition for sample rehydration is around 5 hrs for 50 -100 V depending upon the sample and it continues with the IEF run.
• Components of the rehydration solution:
Urea and Thiourea: Urea solubilizes and denatures proteins, unfolding them to expose internal ionisable amino acids. Commonly 8 M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary for complete sample solubilization. Thiourea, in addition to urea, can be used to further improving protein solubilisation.
Detergent: This solubilizes hydrophobic proteins and minimizes protein aggregation. The detergent must have zero net charge therefore only non-ionic and zwitterionic detergents are used. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to 4% are most commonly used.
Reductant cleaves disulphide bonds to allow proteins to unfold completely. DTT or DTE (20 to 100 mM) are commonly used. 2-Mercaptoethanol is not recommended, because higher concentrations are required, and impurities may result in artefacts. Tributyl phosphine (TBP) is not recommended as reductant for IEF due to its low solubility and poor stability in rehydration solution. Reductants should be added directly before use.
IPG Buffer or Pharmalyte (carrier ampholyte mixtures) improve separations, particularly with high sample loads. Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pH gradient without disturbing IEF or affecting the shape of the gradient. IPG Buffers are carrier ampholyte mixtures specially formulated not to interfere with silver staining following 2-D electrophoresis. Select an IPG buffer with the sample pH interval as the Immobiline DryStrip to be rehydrated
The advantages of increased concentration of IPG Buffer / Pharmalyte are:
- Improved sample solubilization
- Increased tolerance to salt in sample
- A more even conductivity in the gel
The drawbacks of increased concentration of IPG Buffer / Pharmalye are:
- Higher concentrations will limit the voltage use during IEF and increase the time required for the focusing step.
- Silver staining may require a prolonged fixing step to wash out carrier ampholyte that may cause staining background.
IPG Buffer or Pharmalyte can be included in the stock rehydration solution or added just prior to use. (The carrier ampholytes are included in the stock solution when multiple IPG strips of the same pH range are to be used. Carrier ampholytes are added to single aliquots of the stock solution when the same stock solution will be used with different pH range IPG strips).
Tracking dye (bromophenol blue) allows IEF progress to be monitored at the beginning of the protocol. If the tracking dye does not migrate toward the anode, no current is flowing. The dye leaves the strip well before the sample is focused!
Sample can be applied by including it in the rehydration solution. Up to 1 mg of sample per trip can be diluted or dissolved in rehydration solution prior to IEF. The amount of sample required is dictated in part by the detection or visualization method used. Radiolabelling requires a very small amount of sample, silver staining requires typically 1 to 100 μg of sample, and Coomassie™ blue staining and preparative applications require larger sample amounts.
Whether the strips are hydrated actively or passively, it is very important that they be incubated with sample for at least 12 hr prior to focusing. This allows the high molecular weight proteins time to enter the gel after the gel has become fully hydrated and the pores have attained full size. These sample application methods work because IEF is a steady-state technique, so proteins migrate to their pI independent of their initial positions.
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2-D Electrophoresis using immobilized pH gradients: Principles and Methods, Amersham Biosciences, 80-6429-60, Edition AC