Calibrating Protein Molecular Weight

The molecular weight of a protein can be determined based on its relative mobility, by constructing a standard curve using proteins of known molecular weights.

The protein mobility in SDS-PAGE gels is dependent on:

  • Length of the protein in its fully denatured state
  • Extent and types of protein glycosylation
  • SDS-PAGE buffer systems
  • Secondary structure of the protein

Order Novex® Protein Gels

The same molecular weight standard may have slightly different mobility, resulting in different apparent molecular weight when run in different SDS-PAGE buffer systems. If you are using the Novex® protein molecular weight standards, use the apparent molecular masses of these standards in the gels listed in Tables 5 through 8 to determine an apparent molecular weight of your protein.

Apparent molecular masses of Mark 12™ Unstained Standards and Novex® Sharp Prestained Protein Standards under various gel and buffer conditions

Mark 12™ Unstained Standard NuPAGE® (4–12%) Bis-Tris/MES NuPAGE® (4–12%) Bis-Tris/MOPS NuPAGE® (3–8%) Tris-Acetate
Myosin200 kDa200 kDa200 kDa
β-Galactosidase116.3 kDa116.3 kDa116.3 kDa
Phosphorylase B97.4 kDa97.4 kDa97.4 kDa
Bovine serum albumin66.3 kDa66.3 kDa66.3 kDa
Glutamic dehydrogenase55.4 kDa55.4 kDa55.4 kDa
Lactate dehydrogenase36.5 kDa36.5 kDa36.5 kDa
Carbonic anhydrase31 kDa31 kDa31 kDa
Trypsin inhibitor21.5 kDa21.5 kDa21.5 kDa
Lysozyme14.4 kDa14.4 kDa14.4 kDa
Aprotinin6 kDa6 kDaNA
Insulin B chain3.5 kDaNANA
Insulin A chain2.5 kDaNANA
Novex® Sharp Prestained Protein Standard NuPAGE® (4–12%) Bis-Tris/MES NuPAGE® (4–12%) Bis-Tris/MOPS NuPAGE® (3–8%) Tris-Acetate
Band 1260 kDa260 kDa260 kDa
Band 2160 kDa160 kDa160 kDa
Band 3110 kDa110 kDa110 kDa
Band 480 kDa80 kDa80 kDa
Band 560 kDa60 kDa60 kDa
Band 650 kDa50 kDaT50 kDa
Band 740 kDa40 kDa40 kDa
Band 830 kDa30 kDa30 kDa
Band 920 kDa20 kDaNA
Band 1015 kDa15 kDaNA
Band 1110 kDa10 kDaNA
Band 123.5 kDaNANA

Table 1

Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards under various gel and buffer conditions

SeeBlue® Prestained Standard NuPAGE® (4–12%)
Bis-Tris/MES
 NuPAGE® (4–12%)
Bis-Tris/MOPS
NuPAGE® (3–8%)
Tris-Acetate
Myosin188 kDa191 kDa210 kDa
BSA62 kDa64 kDa71 kDa
Glutamic dehydrogenase49 kDa51 kDa55 kDa
Alcohol dehydrogenase38 kDa39 kDa41 kDa
Carbonic anhydrase28 kDa28 kDaNA
Myoglobin18 kDa19 kDaNA
Lysozyme14 kDa14 kDaNA
Aprotinin6 kDaNANA
Insulin3 kDaNANA
SeeBlue® Plus2
Prestained Standard
NuPAGE® (4–12%)
Bis-Tris/MES
NuPAGE® (4–12%)
Bis-Tris/MOPS
NuPAGE® (3–8%)
Tris-Acetate
Myosin188 kDa191 kDa210 kDa
Phosphorylase B98 kDa97 kDa111 kDa
BSA62 kDa64 kDa71 kDa
Glutamic dehydrogenase49 kDa51 kDa55 kDa
Alcohol dehydrogenase38 kDa39 kDa41 kDa
Carbonic anhydrase28 kDa28 kDaNA
Myoglobin17 kDa19 kDaNA
Lysozyme14 kDa14 kDaNA
Aprotinin6 kDaNANA
Insulin3 kDaNANA
Table 2

Apparent molecular masses of Novex® Sharp Prestained Protein Standards and Mark 12™ Unstained Standards on Tris-glycine and Tricine gels

Novex® Sharp Prestained Protein Standard Tris-glycine gels (4–20%) Tricine gels (10–20%)
Band 1260 kDa260 kDa
Band 2160 kDa160 kDa
Band 3110 kDa110 kDa
Band 480 kDa80 kDa
Band 560 kDa60 kDa
Band 650 kDa50 kDa
Band 740 kDa40 kDa
Band 830 kDa30 kDa
Band 920 kDa20 kDa
Band 1015 kDa15 kDa
Band 1110 kDa10 kDa
Band 12NA3.5 kDa
Mark 12™ Unstained Standard Tris-glycine gels (4–20%) Tricine gels (10–20%)
Myosin200 kDa200 kDa
ϐ-Galactosidase116.3 kDa116.3 kDa
Phosphorylase B97.4 kDa97.4 kDa
Bovine serum albumin66.3 kDa66.3 kDa
Glutamic dehydrogenase55.4 kDa55.4 kDa
Lactate dehydrogenase36.5 kDa36.5 kDa
Carbonic anhydrase31 kDa31 kDa
Trypsin inhibitor21.5 kDa21.5 kDa
Lysozyme14.4 kDa14.4 kDa
Aprotinin6 kDa6 kDa
Insulin B chainUnresolved insulin3.5 kDa
Insulin A chain 2.5 kDa
Table 3

Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards on Tris-glycine and Tricine gels

SeeBlue® Prestained Standard Tris-glycine gel (4–20%) Tricine gels (10–20%)
Myosin250 kDa210 kDa
BSA98 kDa78 kDa
Glutamic dehydrogenase64 kDa55 kDa
Alcohol dehydrogenase50 kDa45 kDa
Carbonic anhydrase36 kDa34 kDa
Myoglobin30 kDa23 kDa
Lysozyme16 kDa16 kDa
Aprotinin6 kDa7 kDa
Insulin4 kDa4 kDa
SeeBlue® Plus2 Prestained Standard Tris-glycine gels (4–20%) Tricine gels (10–20%)
Myosin250 kDa210 kDa
Phosphorylase B148 kDa105 kDa
BSA98 kDa78 kDa
Glutamic acid dehydrogenase64 kDa55 kDa
Alcohol dehydrogenase50 kDa45 kDa
Carbonic anhydrase36 kDa34 kDa
Myoglobin22 kDa17 kDa
Lysozyme16 kDa16 kDa
Aprotinin6 kDa7 kDa
Insulin4 kDa4 kDa
Table 4

Protein Secondary Structure

When using SDS-PAGE for molecular weight determination, slight deviations from the calculated molecular weight of a protein (calculated from the known amino acid sequence) can occur due to the retention of varying degrees of secondary structure in the protein, even in the presence of SDS. This phenomenon is observed in highly organized secondary structures (collagens, histones, or highly hydrophobic membrane proteins) and in peptides, where the effect of local secondary structure becomes magnified relative to the total size of the peptide.4 kDa

Buffer Systems

Slight differences in protein mobilities also occur when the same proteins are run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a different pH, which affects the charge of a protein and its binding capacity for SDS. The degree of change in protein mobility is usually small in natural proteins but more pronounced with “atypical” or chemically modified proteins, such as prestained standards.