Very Low Molecular Weight Proteins Electrophoresis Protocol   

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Abstract

The electrophoresis is the most used technique to separate proteins and usually the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proposed by Laemmli is the prefered since it can diferentiate large size proteins, but for very low molecular weight proteins such as 3 KDa and below becomes difficult. Thus a modification of the basic Laemmli SDS-PAGE protocol was required to allow for proteins up to 10 KDa to be separated, maintaining good resolution and reproducible results. This work demonstrates how a 18% gel and modifications of the basic Laemmli protocol acrylamide gel let protein samples with 1 KDa and 0.6 KDa be visible and separated.

Keywords: Electrophoresis, SDS-PAGE, Proteins, Low Molecular Weight, Separation, Purification

Background

Electrophoresis is a technique in which using an electric field separates molecules. “A very common electrophoresis method to separate and to denature proteins which uses a discontinuous polyacrylamide gel as a support medium is called Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The most commonly used protocol is also called the Laemmli method which refers to the first published protocol of this technique established by Laemmli in 1970 (Ornstein, 1964).

The SDS-PAGE can be used to separate proteins, estimate relative molecular mass, determine the relative abundance of major proteins in a sample, and/or determine the distribution of proteins among fractions. Different staining methods can be used to detect rare proteins and to address their biochemical properties. Rath in her work compares the migration of reference proteins at different gel concentration with gels ranging from 11 to 18% not getting a good resolution in molecules under ~6 KDa (Rath et al., 2013; DeWald et al., 1986).

In order to analyze low molecular weight proteins by this electrophoresis system, high concentration gels are needed, but these gels are very brittle and thus difficult to handle. With a modification of the basic Laemmli SDS-PAGE protocol, proteins with 10 KDa or less will be separated maintaining a good resolution with reproducible results. This method will be explained in this work and demonstrated using 1 KDa and 0.6 KDa proteins as samples.

Materials and Reagents

  1. Glass plate
  2. Pre-stained Protein MW marker (Amersham, catalog number: RPU755)
  3. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678)
  4. SDS (Sigma-Aldrich, catalog number: L3771)
  5. Acrylamide-bis (19:1) (Bio-Rad Laboratories, catalog number: 161-0123)
  6. Bromophenol Blue (Sigma-Aldrich, catalog number: B0126)
  7. Tris-base (Promega, catalog number: H5131)
  8. Glycine (Bio-Rad Laboratories, catalog number: 161-0718)
  9. EDTA (Sigma-Aldrich, catalog number: E9884)
  10. Glycerol (Promega, catalog number: H5433)
  11. TEMED (HiMedia, catalog number: RM1572)
  12. Urea (MP Biomedicals, catalog number: 103209)
  13. HCl 37% (Riedel de Haen, catalog number, 30721-2.5L-GL)
  14. Resolution Buffer (Lower) 4x (see Recipes)
  15. Stacking Buffer (Upper) 4x (see Recipes)
  16. 10x Running buffer (see Recipes)
  17. Sample buffer (see Recipes)

Equipment

  1. 1 ml pipette
  2. Protein mini gel cassettes (Bio-Rad Laboratories, catalog number: 1658000FC)
  3. Power Supply (Bio-Rad Laboratories, catalog number: 1645070)

Procedure

  1. For the Gel preparation after cleaning the glass plates, assemble the cassettes following the manufacturer’s instructions. Using the recipe below (Table 1) the two gels are mixed, first the separation gel according to the order described and then the stacking gel. To avoid polymerization, after adding TEMED, mix well and quickly transfer the gel solution by using a 1 ml pipette to the casting chamber between the glass plates and fill up to about 6 cm from bottom to top. Then, prior to polymerization add dH2O to straighten the level of the gel. After polymerization remove the water, prepare the stacking gel and follow the same steps, prior to polymerization insert the appropriate comb until polymerization.

    Table 1. 18% Polyacrylamide gel recipe for very low molecular weight protein


    As soon as the gel is polymerized it can be mounted into an electrophoresis tank, and then filled with running buffer between the gel cassettes and the outside region. 
  2. Once the electrophoresis tank is connected to the power supply, perform a pre-run for 15 min at 20 A. After the pre-run, load the prepared samples into the different wells of the gel, approximately 10 µl of the samples are loaded with sample buffer (5 µl of sample and 5 µl of sample buffer without heating) and 5 µl of the MW marker. The run will be held for 45 min under constant current at 25 A. 
  3. The samples used here are microcystins, a hepatotoxin produced by cyanobacteria with a theoretical weight of 0.995 KDa and Bromophenol blue with a theoretical weight of 0.669 KDa (Reference 3). After the run, stain the gel with Coomassie Blue. The resulting run (Figure 1) shows a clear difference between the microcystin samples and the bromophenol blue marker.
  4. If the mass of protein of interest present in the sample is about 0.2 μg or more, the Coomassie blue staining is typically used. Otherwise, silver staining can be an alternative, as it is more sensitive and can detect masses of proteins as little as 5 ng.


    Figure 1. Polyacrylamide gel. On lane 1 molecular weight marker, lane 2 Bromophenol Blue, lane 3 white, lane 4 sample 1, lane 5 sample 2.

Recipes

  1. Resolution Buffer (Lower) 4x
    Tris-base
    181.71 g (1.5 M) (Adjust pH to 8.8 with HCl)
    SDS
    4 g (0.4%)
    dH2O
    c.s.p.1 L
  2. Stacking Buffer (Upper) 4x
    Tris-base
    60.58 g (0.5 M) (Adjust pH to 6.8 with HCl)
    SDS
    4 g (0.4%)
    dH2O
    c.s.p.1 L
  3. Running Buffer 10x
    Glycine
    1.92 M
    Tris-HCl
    0.25 M
    SDS 1%
  4. Protein samples Buffer 4x
    Tris-HCl
    200 mM, pH 6.8
    EDTA  400 mM
    SDS 8%
    Glycerol 40%

Acknowledgments

This work was possible thanks to the IDEA foundation, the ecology and aquatic system’s laboratory from the Venezuela´s Central University, and is a modification of Laemmli (1970).
  We wish to thank the anonymous reviewers and to Carlos Cáceres and Ysvic Inojosa for his comments and corrections on the manuscript.
  Competing interests: The author declares no conflict of interest.

References

  1. DeWald, D. B., Adams, L. D. and Pearson, J. D. (1986). A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000. Anal Biochem 154(2): 502-508.
  2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  3. National Center for Biotechnology Information. PubChem Compound Database; CID=8272, https://pubchem.ncbi.nlm.nih.gov/compound/8272 (accessed Sep. 25, 2018).
  4. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121: 321-349.
  5. Rath, A., Cunningham, F. and Deber, C. M. (2013). Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts. Proc Natl Acad Sci U S A 110(39): 15668-15673.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rivera, C. E., Rosales, J. D., Freites-Perez, J. C. and Rodriguez, E. (2018). Very Low Molecular Weight Proteins Electrophoresis Protocol. Bio-protocol Bio101: e3093. DOI: 10.21769/BioProtoc.3093.
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