How to Select the Correct MWCO for DNA, RNA and Protein Purification and Concentration

How to Select the Correct MWCO for DNA, RNA and Protein Purification and Concentration

What is MWCO?

Molecular weight cut-off (MWCO) is a term in membrane filtration to describe the pore size or rejection capability of an ultrafiltration membrane. The MWCO of an ultrafiltration membrane is a nominal rating based on its ability to retain > 90% of a known molecular weight of a test molecule, typically non-ionic solutes such as polyethylene glycol (PEG), dextrans and various proteins different molecular weights.

MWCO is measured in Daltons(Da) or kiloDaltons(kDa), and 1 kDa equaling 1000 Da. A single hydrogen atom possesses a mass of 1 Dalton (Da), while the molecular weights of proteins and other macromolecules are commonly measured in kilodaltons (kDa) or simply kD.

Here recommended that the MWCO of the selected ultrafiltration membrane should be 1/3 to 1/2 of the target molecule’s molecular weight for optimal recovery of most macromolecules.

How does MWCO impact separation?

Choosing a membrane with low molecular weight cutoff (MWCO) can improve recovery rates but may simultaneously reduce the speed of ultrafiltration, and lead to the retention of lower molecular weight contaminants.

Conversely, choosing a larger MWCO membrane which means greater surface areas, can potentially lead to increased non-specific binding and permeation.

To ensure efficiently separation, it is crucial to choose an appropriate molecular weight cut-off (MWCO) membrane.

Once sample volume is determined, the most important step is to identify the molecular weight of the sample molecules. Which is essential for selecting the appropriate molecular weight cut-off(MWCO).

How to choose right MWCO for DNA, RNA and Protein?

MWCO Selection for RNA

The average molecular mass of ribonucleotide is 339.5 Da, but when nucleotides combine to form ribonucleic acid(RNA), one water molecule is removed, resulting in the average molecular weight of ribonucleotide in RNA should be calculated according to 321.5 Da. Assuming that our target mRNA consists of n ribonucleotides, its molecular mass can be calculated as 321.5*n Da. According to the pore size of the ultrafiltration membrane should be 1/3 of the molecular mass of the target mRNA, the MWCO of the ultrafiltration filter should be 107*n Da.

For example, let’s consider a scenario when the length of the target mRNA is 1000 nt. Its molecular weight is 321.5*1000 Da=321,500 Da=321.5 kDa, and it needs to be retained by an ultrafiltration membrane with MWCO of 107 kDa.

By applying the following formula in reverse, we can get different MWCO specifications corresponding to the minimum length of the target mRNA (unit: nt), which also means, the minimum number of ribonucleotides the target mRNA should contain.

For instance, let’s calculate the minimum mRNA length required to be retained by an ultrafiltration membrane with MWCO of 100 kDa, using the following formula:

 formula

This means as long as our mRNA consists of more than 935 ribonucleotides, we can effectively use a 100 kDa centrifugal filter to intercept it.

Applying the calculation logic outline above, we can come up with the following table:

Minimum nucleic acid length(nt)

Size of ultrafiltration centrifuge tubes(kDa)

28

3

94

10

281

30

935

100

 

MWCO Selection for DNA

In a similar manner, the average molecular weight of deoxyribonucleotides is 327 Da, and one molecule of water is removed during the formation of deoxyribonucleic acid(DNA). Consequently, the average molecular weight of deoxyribonucleotides in DNA is calculated as 309 Da.

Assuming our target DNA strand consists of n deoxyribonucleotides, its molecular weight is 309*n Da. According to the MWCO of the ultrafiltration centrifugal filter should be 1/3 of the target DNA’s molecular weight, the MWCO of the chosen centrifugal filter should be 103*n Da.

For instance, a DNA strand with a length of 1000 nt has a molecular weight of 309*1000 Da = 309,000 Da = 309 kDa, which needs to be retained by an ultrafiltration membrane with a MWCO of 103 kDa.

Using the same principles, we can get the following table:

Minimum nucleic acid length(nt)

Size of ultrafiltration centrifuge tubes(kDa)

30

3

97

10

292

30

971

100

MWCO Selection for Protein

The average molecular weight of an amino acid is approximately 128 Da, and when one molecule of water is removed to form a protein, the average molecular weight of an amino acid residue in a peptide chain becomes approximately 110 Da.

As an example, for immunoglobulin IgG, each of the two heavy chains typically consists of around 450-550 amino acid residues (use 500 for calculation), and each of the two light chains comprises approximately 214 amino acid residues. Consequently, the molecular weight of IgG can be estimated at approximately 1420*110 Da, which equals to about 156 kDa.

Here are some common protein molecular weights and corresponding MWCO to consider:

MW concentration

MW(Da)

MWCO

Chymotrypsinogen(1mg/mL)

25,000

3K

Cytochrome C(0.25mg/mL)

12,400

3K

Lysozyme(0.25mg/mL)

14,400

3K

Ovalbumin(1mg/mL)

45,000

10K

Chymotrypsinogen(1mg/mL)

25,000

10K

BSA(1mg/ml)

67,000

30K

Ovalbumin(1mg/ml)

45,000

30K

Thyroglobulin(0.5mg/ml)

677,000

100K

IgG(1mg/ml)

156,000

100K

This method is not absolute because the shape and structure of proteins can be complicated. However, it serves as a practical approach for pre-screening and selecting membranes for a specific application. The following rules of thumb are useful for separation:

1. To separate two solutes with different molecular weights, the molecular masses should differ by a factor of 10.

2. The MWCO of the membrane must be at least 1/2 of the solute’s molecular weight to be retained.

3. Molecule shape impact separations. When working with approximately globular molecules, it is best to initially test a centrifugal filter with MWCO equal to or slightly smaller than the molecular weight of the target molecule.

4. When working with samples without reference, it is advisable to begin by selecting a centrifugal filter with a molecular weight cut-off (MWCO) that is half the molecular weight of the target molecule. Choose centrifugal filter units here.

Want to understand how factors such as sample concentration, transmembrane pressure, molecules composition and more affect the retention capability? Read this article: How to Choose MWCO Filters?