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Interpretation of Molecular Sieve Results

Size exclusion chromatography (SEC) is also called gel chromatography, commonly
known as molecular sieves. When the eluent is an aqueous solution or buffer, it is
called gel filtration chromatography (GFC), which is widely used in the field of biology;
when an organic solvent is used as the eluent, it is called gel permeation
chromatography (GPC), which is widely used in polymer field. Size exclusion
chromatography is commonly used to separate high molecular weight compounds
such as tissue extracts, peptides, proteins and nucleic acids.

Size exclusion chromatography separates samples based on differences in protein
molecular weight or molecular shape. As the sample moves down from the top of the
column, large protein molecules cannot enter the gel particles and are eluted quickly;
while smaller protein molecules can enter the gel particles, and the smaller proteins
enter the gel with different retention times. The larger the molecular weight, the earlier
the elution completed, allowing proteins with different molecular sizes to be separated.

Figure 1. Principle of Size Exclusion Chromatography (Wolf et al, 2015)

Molecular sieves are also commonly used in protein separation and purification.
There are two main functions, one is separation and purification; the other is protein
analysis. Newbies might not know how to analyze molecular sieve results when they 
first come into contact with them. Let’s use different examples to explain some of
their differences.

Separation and Purification

In scientific research experiments, usually the first step of protein purification is affinity
chromatography, but in most cases, it is difficult to achieve high purity with one-step
chromatography. According to the SDS-PAGE gel pattern results, molecular sieves can
be used to further separate and purify the target protein. What needs to be paid
attention to is the selection of molecular sieve specifications. The column specifications
used for proteins of different sizes may be different. The selection needs to be combined
with the resolution of the molecular sieve column. The column manual can be really
helpful if you are not familiar with it.

Figure 2. Nickel column purification results
(Lane 8 and 9 are Elution results)

Figure 3. Lane 8/9 through molecular sieve

Figure 4. Molecular sieve electrophoresis results

It can be seen from the results that molecular sieve can effectively separate impurity
proteins and target proteins. However, it should be noted that when using molecular
sieves to separate and purify target proteins, there should be a large difference in
molecular weight (more than double the molecular weight) for effective separation.

Protein Analysis Function

Another role of molecular sieves is as a protein analysis tool. The molecular weights
of multi-aggregated proteins and monomeric proteins are different, and the molecular
weights of complexes and single subunits are also different. Molecular sieves can use
this difference in molecular weight to analyze and separate protein states.

Figure 5. Protein analysis results
(Lane 1 is an affinity chromatography sample with high purity. The molecular sieve has two peaks and the main
peak is obvious. It may be a dimer or a polymer, and the sample has good uniformity)

Figure 6. Protein analysis results

The purity of the protein sample after affinity chromatography purification is high.
Two obvious peaks appear in molecular sieve analysis. The size of the protein detected
by SDS-pag gel is consistent. It can be seen that the homogeneity of the protein after
purification is not high. Polymers and monomers are mixed. Through Molecules can
separate proteins in different states, and subsequent work can be carried out using
proteins in different states as needed.

Journal reference:

1.Schmidt TG & Skerra A (1994) J Chromatogr A., 676, 337-45.
2.Schmidt TG, Koepke J, Frank R & Skerra A (1996) J Mol Biol., 255, 753-66.
3.Voss A & Skerra (1997) Protein Eng., 10, 975-82.
4.Knabel M, Franz TJ, Schiemann M, Wulf A, Villmow B, Schmidt B, Bernhard H, Wagner H & Busch DH (2002) Nat Med., 8, 631-7
5.Junttila MR, Saarinen S, Schmidt T, Kast J, Westermarck J (2005) Proteomics, 5, 1199-203.
6. Schmidt TG, Batz L, Bonet L, Carl U, Holzapfel G, Kiem K, Matulewicz K, Niermeier D, Schuchardt I & Stanar K (2013) Protein Expr Purif., 92, 54-61.
7.Ivanov KI, Bašić M, Varjosalo M & Mäkinen K (2014) JoVE, 86, 51536.
8.Carl U (2016) Poster session presented at: PEGS Bosten 2016.
9.Dintner S, Heermann R, Fang C, Jung K & Gebhard S (2014) J Biol Chem., 289, 27899-910.
10.Yeliseev A, Zoubak L, Schmidt TGM (2017). Protein Expr Purif., 131, 109-118.

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