Very Low Molecular Weight Proteins Electrophoresis Protocol ^Updated   

Abstract

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.