Rehydration buffer

Pour 300-400 pi of rehydration buffer D into a small tray. Peel off the protective foil and insert the IPG gel face down into the tray. Cover gel and rehydration buffer with 1 - 2 ml silicone oil, close the lid of the tray, and allow swelling overnight at RT by gentle agitation. 

Use a silicone rubber frame (sample applicator) or a piece of filter paper (2x5 mm) for sample application about 5 mm from die anode or cathode and apply sample (concentration should not exceed 10 mg/ml), which is dissolved in rehydration buffer. 

The part is transferred into the groove containing 80 pL of rehydration buffer and incubated for 5 min. 

Using the special 25-ml syringe included with the kit, fill the cassette with rehydration buffer by injecting from the bottom until the top of the gels are just... 

Remove the rehydration buffer from the lEF strips and save at 4°C for future re-use (see Note 6). 

If the final concentrations vary, dilute the more concentrated samples with lysis buffer before labeling to ensure equivalent labeling in all reactions. After labeling, the combined sample is diluted with 2x sample buffer, and the volume increased with standard 2-D rehydration buffer to the volume required for the specified ImmobilineT DryStrip (IPGstrip). 

Make up protein samples [80-100 pg of radioactively labeled proteins, 3 x 50 pg of DIGE-labeled proteins (see Note 5), or 350-600 pg of unlabeled proteins for Colloidal Coomassie staining) (see Note 6)] to 360 pL with 8 M urea/2 M thiourea. Subsequently, add 40 pL of lOx rehydration buffer and mix the solution by shaking at room temperature for 30 min. Centrifuge the rehydration mix for 5 min at 21,000 x g (20 °C) to remove insoluble proteins. 

Place the gel slice in an Eppendorf tube and add sufficient rehydration buffer (solution 3) to immerse. 

Notes HeLa cells (1 x 106) were formalin-fixed in an equal volume of 1% agarose. After histological processing and paraffin embedding, the cell plugs were rehydrated and resuspended in the indicated buffer. Total protein in the supernatants was assessed colorimetrically after heating at the indicated temperatures and times. The % recovery values are the mean, the standard deviation and relative to a fresh cell lysate from the sample number of cells (for more detail, see Reference 25). 

It is possible to restore antigenicity in tissue sections by digesting the rehydrated sections with any of several proteolytic enzymes, or by incubating the tissue section at elevated temperatures in any of several liquids, including water and various buffers (antigen retrieval).12... 

Figure 19.2 A generalized proteomics work flow for the extraction and identification of proteins in FFPE tissue. Formalin-fixed tissues acquired by sectioning, needle dissection, or laser capture are deparaffinized in xylenes and are rehydrated in graded alcohols. The material is resuspended in buffer which generally contains a detergent/ protein denaturant and the sample is heated to complete the extraction process. The protein extract is reduced, alkylated, and digested with trypsin before protein profiling.

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

Transfer the deparaffmized, rehydrated, and methanolic-peroxide blocked (optional) slides (see Notes 2-6) into plastic Coplin jars or containers filled with HIER buffer (see Note 7). (See Chapter 12 for preparation of tissue sections.)... 

Place the deparaffmized, rehydrated slides in metal slide racks and immerse in the hot buffer (see Notes 2-6 and 8). Seal the PC and bring to full pressure. Full pressure is attained when both the rise-n-time indicator and the safety plug are in the upright position. Treat the sections at full pressure for 2-3 min. 

Place the deparaffinized, rehydrated slides in metal or heat-resistant plastic slide racks and immerse in an autoclaveable incubation container filled with 250 mL HIER retrieval buffer (see Notes 2-7). Cover the container with a lid to avoid evaporation (see Note 10). 

Some or all prehybridization steps are optional, depending on the target and specimen. Prehybridization entails soaking the slides containing the specimens in a prehybridizafion buffer in order to have the specimen rehydrate and equilibrate with the hybridization buffer, to be used later. The prehybridization phase may also entail treatment with proteases or nucleases in order to improve probe access and to reduce background. 

Air-dryed frozen sections have to be rehydrated in Tris-buffered saline (TBS)... 

Semiaqueous or Nonaqueous Solutions. Although the measurement of pH in mixed solvents (e.g., water/organic solvent) is not recommended, for a solution containing more than 5% water, the classical definition of a pH measurement may still apply. In nonaqueous solution, only relative pH values can be obtained. Measurements taken in nonaqueous or partly aqueous solutions require the electrode to be frequently rehydrated (i.e soaked in water or an acidic buffer). Between measurements and after use with a nonaqueous solvent (which is immiscible with water), the electrode should first be rinsed with a solvent, which is miscible with water as well as the analyte solvent, then rinsed with water. Another potential problem with this type of medium is the risk of precipitation of the KC1 electrolyte in the junction between the reference electrode and the measuring solution. To minimize this problem, the reference electrolyte and the sample solution should be matched for mobility and solubility. For example, LiCl in ethanol or LiCl in acetic acid are often used as the reference electrode electrolyte for nonaqueous measurements. 

If the nitrocellulose has been dried, rehydrate it by incubation for a few minutes in an appropriate volume of TSM buffer (see previous step)... 

The composition of the sample solution should be as similar as possible to the composition of the rehydration solution. Buffers and salts should not be present at a concentration >50 mM. 

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