Experiments on the solubilization of proteins by descaling and leaving agents prior to bidirectional electrophoresis
Because of its outstanding ability to separate total proteins, bidirectional gel electrophoresis can be applied to the separation of all types of proteins. However, the solubility of proteins is a serious obstacle to the analysis of many types of proteins, especially membrane proteins. The source of this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillement, M. Zivi, C. Damerweiler, V. Mitschine, eds.
Operation method
Solubilization of proteins by descaling and leaving agents prior to bidirectional electrophoresis
Materials and Instruments
Protein Move 3.1 Solubilization of protein samples for IEF in urea For more product details, please visit Aladdin Scientific website.
Dodecyl Maltoside Cationic Descaler Urea Storage Solution Urea-Thiourea Storage Solution
Ultracentrifuge IPGphor unit
1. Solubilization of solid samples (e.g. tissue or cell pellets)
In this case, the volume of the sample is often ignored in the terminal dissolution volume. The sample solution contains urea (final concentration 9-9.5 mol/L; see Note 1), the selected descaling agent (according to the list in 2.3.1) at a final concentration of 2%-4% (m/V), an amphoteric electrolyte [ IPG: 0.4% (w/v); [CA] - IEF: 2% (w/v)], a reducing agent (50 mmol/L DTT or 5 mmol/L TBP or 5 mmol/L TCEP), and an amphoteric electrolyte. The sample solution was dissolved by adding it to the solid sample, and ultrasonication for 30 min under aqueous conditions would help to dissolve the proteins, and the insoluble material was removed by centrifugation at high speed for 20,000 g for 30 min.
2. Dissolution of protein samples from suspensions or solutions
In this case, the volume of the sample must be taken into account. Therefore, it is necessary to calculate the final volume. As a rule of thumb, the sample volume can be 35% of the final extraction volume. Solid urea, water and descaler storage solution, and amphoteric electrolytes are added together to the protein sample to dissolve the protein.
3.2 Solubilization of protein samples in urea thiourea
A defined volume of protein sample is hydrated into the IPG tape, or in the case of homemade IPG tapes, made wider than commercial products, and the protein sample can be adjusted to a larger volume (up to 1 ml), which allows dilution of the concentrated dissociative agent and the protein sample. If the descaler can be pre-dissolved in the concentrated ionizer, which also contains the reducing agent, then the sample volume is 2% of the total dissolved volume, and if the descaler has to be added last and the concentration of urea does not exceed 8 mol/L, it is convenient to use 1 part of the sample, 1 part of the descaler reservoir, and 8 parts of the concentrated ionizer. If this method makes the volume too large, it can be solved in two ways:
( 1 ) Add the equivalent of 1 portion of sample descaler to a test tube, concentrate in a vacuum concentrator and then add 1 portion of sample and 4 portions of concentrated leachate.
( 2 ) Considering that the sample volume is 40% of the final volume, the corresponding amounts of urea, thiourea and solid descaler should be weighed and dissolved ultrasonically under ice bath conditions. In all cases, the protein samples were dissolved at room temperature for 30-60 min and then centrifuged at 200,000 g for 30 min to remove insoluble material.
3.3 Protein sample solubilization for zone electrophoresis
Dissolution of protein samples with SDS is not described in this chapter, which focuses on the dissolution of protein samples in urea and cationic detergents prior to non-diagonal electrophoresis [ 16, 17]. Calculating the composition of the samples in the dissolution solution was simple, with the protein samples comprising 1/4 of the final dissolution volume and the liquid samples being added in the following order: 0.4 vol. of 20% (w/v) cationic descaler reservoir, 0.2 vol. of reducing agent, 0.4 vol. of phosphate buffer, and 2 vol. of 8 mol/L urea.
The mixed sample solution was sonicated in a water bath for 30 min and then centrifuged at 10,000 g for 15 min to remove insoluble material.