Two-dimensional gel electrophoresis (2DE) is a powerful technique for separating complex protein mixtures based on their molecular weight and isoelectric point. This technique is widely used in proteomics research to identify and quantify differentially expressed proteins in biological samples.
Serum or plasma samples are commonly used in proteomic studies because they are easily accessible and contain a diverse range of proteins. However, the presence of high-abundance proteins such as albumin and immunoglobulins can interfere with the separation and detection of low-abundance proteins by 2DE. Therefore, it is important to remove or deplete these high-abundance proteins prior to 2DE analysis.
Several methods are available for high-abundance protein removal, including immunoaffinity depletion, chromatography, and ultracentrifugation. Immunoaffinity depletion involves the use of antibodies specific to high-abundance proteins to selectively remove them from the sample. Chromatography methods such as ion-exchange chromatography and size-exclusion chromatography can also be used to remove high-abundance proteins.
Once the high-abundance proteins are removed, the remaining proteins in the sample must be solubilized and prepared for 2DE. The protein extraction and solubilization steps can significantly impact the quality of the 2DE results. Commonly used solubilization methods include the use of chaotropic agents, detergents, and reducing agents.
After solubilization, the protein sample is typically separated by isoelectric focusing (IEF) in the first dimension, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. The resulting protein spots on the 2D gel can then be visualized and analyzed by various techniques, such as staining, western blotting, and mass spectrometry.
1 Affinity Depletion of Serum Albumin
1.1 Batch Use of Dye-Agarose Affinity Resins
Here we describe a generic method, based on manufacturer's instructions, adaptable for the majority of available dye resin slurries. The steps involved are equilibration, binding, washing, and stripping.
A. Add 250 μL of resin to a 1.5-mL microcentrifuge tube, centrifuge for 2 min at 0.2g, and remove liquid using a gel-loading pipet tip.
B. Add 200 μL of 20 mM sodium phosphate, pH 7.0, and shake gently for 10 min. Centrifuge at 1000 rpm for 2 min and remove liquid. Repeat the equilibration twice.
C. Take 25 μL of clarified plasma/serum, dilute it with 175 μL of the 20 mM sodium phosphate, pH 7.0 buffer, and mix thoroughly.
D. Add the diluted plasma to the conditioned resin, vortex briefly, and shake gently for 10 min to allow serum albumin to bind to the dye resin. Centrifuge for 2 min at 1000 rpm and remove supernatant using a gel-loading pipet tip. Transfer solution, containing unbound albumin-depleted proteins, to a clean 1.5-mL microcentrifuge tube.
E. Wash the resin three times with 150 μL 20 mM sodium phosphate, pH 7.0 buffer, shake for 10 min, centrifuge for 2 min at 1000 rpm, remove the supernatant, and combine with the previous solution.
F. Elute the proteins bound to the resin by washing three times with lysis buffer (see Note 3). Combine stripped fractions into a separate 1.5-mL tube.
G. Concentrate the albumin-depleted fraction (approx vol 650 μL) to a final vol of approx. 100 μL using a 10,000 MWCO (molecular weight cut-off) membrane filter. Desalt and prepare for 2-DE by TCA/acetone precipitation (see Note 4). For the albumin-rich fraction, remove a 150-μL aliquot from the stock solution and perform TCA/acetone precipitation directly.
2 Lectin Affinity Serum Albumin Removal Using WGA/Agarose
A. Prepare the Handee Mini Spin column for use by ensuring the frit at the bottom of the main chamber is firmly in place by pushing down with a paper clip. Resuspend the lectin in the buffer supplied by the manufacturer to give a homogeneous lectin slurry, then pipet 200 μL into the chamber, centrifuge briefly, and remove liquid.
B. Add 500 μL of phosphate buffer and shake gently for 10 min. Centrifuge at 1000 rpm for 2 min, remove liquid, and repeat twice.
C. Prepare plasma by diluting 50 μL of plasma with 750 μL of phosphate buffer containing 0.05% SDS. This amount will typically yield 50 μg of albumin-free protein.
D. Transfer diluted plasma to the column containing the beads. Shake gently using a rotary mixer, ensuring good mixing, for 5 min. Centrifuge briefly and collect eluent. Wash beads three times with 200 μL of phosphate buffer. Combine wash eluents (albumin-rich fraction).
E. Elute the glycosylated proteins with 200 μL sugar solution, shaking for 5 min on a rotary shaker. Wash three times with 150 μL of sugar solution and combine, total volume approx 650 μL.
F. Reduce the albumin-depleted fraction to a final vol of approx 100 μL using a 10,000 MWCO membrane filter. Desalt and prepare for 2-DE by TCA/acetone precipitation.
3 Antibody Serum Albumin/Immunoglobulin G Removal
3.1 Use of Antibody/Protein G Resin
A. Pipet 20 μL of plasma/serum into a 1.5-mL microcentrifuge tube and add 750 μL of resin slurry. Close cap and mix on a rotary shaker, ensuring strong mixing is taking place, for 30 min at room temperature.
B. Transfer entire slurry to the upper chamber of a microspin column and insert column into a suitable microcentrifuge tube. Centrifuge at 1000 rpm for 5 min. Remove liquid (approx. 500 μL) and desalt/concentrate.
3.2 Use of Antibody/Protein G Resin
A. Connect a syringe to the anti-SA cartridge using a needle-port adapter and equilibrate packing material with at least five cartridge volumes of PBS solution, pH 7.4.
B. Dilute the serum/plasma 10-fold with PBS solution and inject an amount less than the maximum binding capacity into the cartridge at a steady flow rate.
C. Collect the flowthrough in a clean 0.5-mL microcentrifuge tube. Wash cartridge with three column vols of PBS solution and combine wash fractions in a separate tube.
D. Connect the needle-port adapter to the protein G cartridge and condition the packing material with at least five cartridge vols of PBS solution, pH 7.4.
E. Inject the albumin-depleted fraction from step C at a steady flow rate and collect the flowthrough in a fresh 1.5-mL tube.
F. Inject the combined wash fractions from step C at a steady flow rate and combine with liquid from step F.
G. Concentrate the albumin-IgG-depleted fraction to a final volume of approx 100 μL using a 10,000 MWCO membrane filter. Desalt and prepare for 2-DE by TCA/acetone precipitation.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.