2-D Clean-Up Kit is designed to prepare samples that would otherwise produce poor 2-D results due to high conductivity, high levels of interfering substances, or low concentration of protein.
Current methods of protein precipitation suffer from several significant disadvantages:
2-D Clean-Up Kit circumvents these disadvantages by providing a method for selectively precipitating protein for 2-D electrophoresis. Protein can be quantitatively precipitated from a variety of sources without interference from detergents, chaotropes, and other common reagents used to solubilize protein. Recovery is generally greater than 90%. The procedure does not result in spot gain or loss, or changes in spot position relative to untreated samples. The precipitated proteins are easily resuspended in 2-D sample solution. The procedure can be completed in less than one hour.
The overall quality of protein separation using 2-D Clean-Up Kit has been shown to be superior to that of samples prepared by precipitation with acetone (54). Preparation of protein samples with the kit reduces horizontal streaking, improves spot resolution, and increases the number of spots detected compared with samples treated by other means (Figure 1 and Table 1).
Figure 1. 2-D Clean-Up Kit eliminates horizontal streaking caused by residual SDS. Sample: Rat liver extracted with 4% SDS, 40 mM Tris base. First dimension: Approximately 20 μg rat liver protein, 7-cm Immobiline DryStrip pH 4–7, Ettan IPGphor Isoelectric Focusing System 17.5 kVh. Second dimension: SDS-PAGE (12.5%), SE 260 (8 × 9 cm gel). Stain: Silver Staining Kit, Protein.
* Protein spots were detected using ImageMaster™ 2D Elite software.
† 9.8 M urea, 2% CHAPS, 0.5% IPG Buffer pH 3–10, 65 mM DTT.
The 2-D Clean-Up Kit procedure uses a combination of a unique precipitant and co-precipitant to quantitatively precipitate the sample proteins while leaving interfering substances behind in the solution. The proteins are pelleted by centrifugation and the precipitate is washed to further remove non-protein contaminants. The mixture is centrifuged again and the resultant pellet can be easily resuspended into a 2-D sample solution of choice, compatible with first dimension IEF.
The kit contains sufficient reagents to process 50 samples of up to 100 μl each. The procedure can be scaled-up for larger volumes or more dilute samples.
Precipitant, co-precipitant, wash buffer, wash additive.
Required but not provided
Ice bath, 1.5-mL capped microcentrifuge tubes, microcentrifuge capable of at least 12 000 × g, rehydration solution or IEF sample solution for resuspension (see next section), vortex mixer.
Procedure A is applicable for sample volumes of 1–100 μl containing 1–100 μg of protein. For larger samples containing more than 100 μg of protein, use procedure B.
Prior to starting the procedure, chill the wash buffer to -20 °C for at least 1 h.
Procedure A: For sample volumes of 1–100 μl (containing 1–100 μg of protein per sample)
Process the protein samples in 1.5-mL microcentrifuge tubes. All steps should be carried out on ice unless otherwise specified.
Procedure B: For larger samples of more than 100 μg of protein
All steps should be carried out on ice unless otherwise specified.
Resuspension of pellet
2-D Clean-Up Kit produces a protein pellet. When using cup loading, resuspend the pellet in sample preparation solution (see appendix I). When using rehydration loading, resuspend the pellet in rehydration solution (see options 1 and 2 below), which is applied directly to the Immobiline DryStrip gel.
Sample resuspension volumes
The volume of rehydration solution used to resuspend the sample depends on the sample loading method and the length of the Immobiline DryStrip gel used for the first-dimension separation. If using Ettan IPGphor 3 and the sample is to be loaded onto the Immobiline DryStrip gel using a sample cup, the sample volume should not exceed 150 μl. If the sample is to be loaded onto the Immobiline DryStrip gel by rehydration, the sample volumes shown in Table 2 should be used according to the length of the Immobiline DryStrip gel.
The optimal quantity of protein to load varies widely depending on factors such as sample complexity, the length and pH range of the Immobiline DryStrip gel, and the method of visualizing the 2-D gel separation. General guidelines are given in chapter 2.
The protein concentration of the sample is best determined using the 2-D Quant Kit, which can accurately quantitate protein in the presence of detergents, reductants, and other reagents used in sample preparation.
* DTT is added just prior to use: 7 mg DTT per 2.5-mL aliquot of rehydration stock solution. For rehydration loading, sample is also
added to the aliquot of rehydration solution just prior to use.
† If necessary, the concentration of urea can be increased to 9 M or 9.8 M.
‡ Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octylglucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
§ As an alternative to IPG Buffer, use Pharmalyte 3–10 for Immobiline DryStrip 3–10 or 3–10 NL, Pharmalyte 5–8 for Immobiline
¶ A Pharmalyte/IPG Buffer concentration of 0.5% (125 μl) is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an IPG Buffer/Pharmalyte concentration of 2% (500 μl) is recommended with the Multiphor II and Immobiline DryStrip Kit system.
Store in 2.5-mL aliquots at -20 °C.
* DTT is added just prior to use: Add 7 mg DTT per 2.5-mL aliquot of rehydration stock solution.
† Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octylglucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
‡ A Pharmalyte/IPG Buffer concentration of 0.5% (125 μL) is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an IPG Buffer/Pharmalyte concentration of 2% (500 μL) is recommended with the Multiphor II and Immobiline DryStrip Kit system.
Store in 2.5-mL aliquots at -20 °C.