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Study Notes: Measuring Molecular Weight

Remember that molecular weight is actually a ratio called relative molecular mass (Mr).

Protein bands detected on the gel need to be identified. Identification involves the following factors.

  • Measurement of the molecular weight (Mr)
  • Knowledge and experience with these particular samples and proteins
  • Immunological techniques such as western blotting to identify proteins (Step 7)
  • Purification and analysis of the protein such as amino acid sequencing and comparison with published protein sequence data
  • Other analytical techniques.

Measurement of the Mr is simple and can be carried out on the gel. Remember that in SDS-PAGE the effect of the SDS is to impart a uniform negative charge on the protein based on the size of the protein in a linear way. That is the charge:mass ratios for all proteins are the same. In this case relative mobilities of proteins is based solely on the sieving action of the gel.

The simplest way to measure Mr is to add a set of standard proteins of known Mr to one or more wells in the gel. These are called molecular weight markers and can be sourced commercially in ranges to suit the % gel being run. They also come pre-stained in a range of colours (Rainbow Markers) if required.

The relative migration (from the top of the gel in cm) of each of the marker proteins is plotted on a graph against Mr to form a standard curve. The Mr of an unknown protein can then be simply calculated from the migration of that protein on the gel.

An example of a set of Low Range Mr Markers that come mixed, pre-diluted and ready to use, is shown below.

Protein
Relative Molecular Mass - Mr
Ovalbumin
43 kd
Alpha-chymotrypsinogen
25.7 kd
Beta-lactoglobulin
18.4 kd
Lysozyme/Cytochrome C
13.3 kd (14.3 and 12.3)
Bovine Trypsin Inhibitor
6.2 kd
Insulin
3.0 kd

The standard curve plotted from SDS-PAGE on a 15% gel.

Calibration chart for Mr standards
Calibration chart for Mr standards.

An example of the determination of the Mr of an unknown protein Px is shown. Px was run on a 15% gel and migrated 9 cm into the gel. Using the standard curve the Mr is calculated to be 9 kd.

An example of the use of Mr markers is given below. This gel shows analyses of virus-infected cells run on an 11% to 24.4% polyacrylamide gradient gel. The Mr markers are shown in the left hand lane and the Mr of six of them is indicated on the right hand side of the gel. Notice the ‘splaying’ of the sample lanes from top to bottom due to the high strength acrylamide used towards the bottom of the gradient.

A photograph of an actual SDS-PAGE gel showing molecular weight markers. A series of bands at the left shows the Mr markers.
An SDS-PAGE separation on a gradient gel. Notice the Mr markers in the far left lane.

 

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