Mass estimation of native proteins by blue native electrophoresis: principles and practical hints

Abstract

Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.

Fig. 1.

Protein migration velocity on acrylamide gradient gels decreases during blue native electrophoresis, but no protein is completely immobilized after commonly used electrophoresis times (<5 h). Black dots on solid black lines mark dodecylmaltoside-solubilized membrane protein complexes: respiratory complex I (I; 1 MDa), monomeric ATP synthase (M; 597 kDa), respiratory complex III (III; 482 kDa), respiratory complex IV (IV; 205 kDa), and respiratory complex II (II; 123 kDa). Red triangles on dashed red lines mark soluble proteins from the high molecular weight kit: thyroglobulin (TG; 669 kDa), ferritin (FER; 476 kDa), catalase (CAT; 239 kDa), and BSA (66 kDa). BSA and catalase that were visible as blue bands during BNE for some hours were no longer detected at later stages. A double headed arrow marks the uniform 3.5% acrylamide layer on top of the 3.5–16% acrylamide gradient gel. Mass/migration data were taken from Table I.

Fig. 2.

Mass calibrations on BN gels by membrane and soluble proteins differ at all electrophoresis times. Dodecylmaltoside-solubilized bovine mitochondrial complexes (lanes B) and soluble proteins of the high molecular weight kit (lanes H) were applied to 3.5–16% acrylamide gradient gels. Assignment of protein bands is as in Fig. 1 and Table I. A–E, following BNE for 2, 3, 4, 5, and 10.5 h, respectively, the gels were fixed and Coomassie-stained. Dashed green lines mark specific acrylamide concentrations in the gradient gels and relate to the actual position of the analyzed proteins. F, regression lines for the calibrations by membrane proteins (dots on solid lines) and soluble proteins (triangles on dashed lines) are indicated for various electrophoresis times (green for gel A (2 h), brown for gel B (3 h), black for gel C (4 h), and blue for gel E (10.5 h). Note the inversion of the slopes for membrane and soluble proteins upon transition from 2 to 3 h of electrophoresis (see “Discussion”). Mass/migration data were taken from supplemental Table S1. CAT, catalase; FER, ferritin; LDH, lactate dehydrogenase; TG, thyroglobulin.

Fig. 3.

Mass estimation of small proteins by BNE. Dodecylmaltoside-solubilized mitochondrial proteins and protein complexes were separated by one-dimensional BNE (A) using a 3.5–16% acrylamide gradient gel with a uniform 3.5% acrylamide layer on top (boxed area). Two-dimensional separation by Tricine-SDS-PAGE revealed the characteristic polypeptide patterns of respiratory complexes I–IV (I, II, III, and IV) and monomeric ATP synthase (M). Several further proteins in the low mass range (protein numbers 1–16) were identified by mass spectrometry (Table II, supplemental Figs. S7–S22, and supplemental Tables S6–S9). Proteins that were monomeric in one-dimensional BNE (protein numbers 1–8) were found in an arclike arrangement. Protein subunits from homo- and heteromeric complexes (protein numbers 7d and 9–16) were dissociated by SDS and found below the arclike structure. B, small membrane proteins (blue dots; protein numbers 4, 8, and 15) and most soluble proteins (small red triangles) fitted the extended regression line for mitochondrial membrane protein complexes I–IV and monomeric ATP synthase (M) in contrast to a few further soluble proteins (large red triangles; protein numbers 5, 11, and 16b). This non-uniform behavior of soluble proteins may relate to a non-uniform binding of Coomassie dye (see “Discussion”). Protein numbers 1–3 (in the 15–20-kDa range) were too small to be separated by the 3.5–16% acrylamide gel used for BNE and comigrated with the Coomassie dye front.

Fig. 4.

Mass calibration by membrane and soluble proteins is almost identical using 3.5–13% acrylamide gradient gels for BNE. A, separation of soluble proteins of the high molecular weight kit. B, separation of TX- and DDM-solubilized mitochondrial complexes from chicken heart (CH) and from BHM. C, separation of digitonin (Di)-solubilized mitochondrial complexes and supercomplexes from chicken and bovine heart. Assignment of proteins and complexes is as in Fig. 1. Additional supercomplexes a–d from BHM comprise respiratory complexes I, III, and IV in different stoichiometric ratio as detailed in supplemental Table S3. PDHC, pyruvate dehydrogenase complex; T and H, tetrameric and hexameric forms of ATP synthase; CAT, catalase; FER, ferritin; LDH, lactate dehydrogenase; TG, thyroglobulin. Bands 1–7 from chicken heart show almost identical migration as bovine complexes IV, III, monomeric ATP synthase (M), I, and supercomplexes b–d, the bovine counterparts. D, mass calibration by soluble proteins of the high molecular weight kit (red triangles) and by DDM-solubilized complexes from bovine (black dots) and chicken heart (green circles). E, mass calibration by soluble proteins (red triangles) and by digitonin-solubilized complexes from bovine (black dots) and chicken heart (green circles). Mass/migration data were taken from supplemental Table S3. Regression lines for calibration by membrane and soluble proteins were almost parallel and close together. A conversion factor of 0.8 was required to estimate the masses of DDM-solubilized membrane proteins after calibration by soluble proteins of the high molecular weight kit (see D). Similarly, a conversion factor around 0.7 was required for digitonin-solubilized membrane proteins (see E). D, dimeric ATP synthase.

Fig. 5.

Mass calibration by membrane and soluble proteins on 3–12% acrylamide gradient gels for hrCNE and CNE. A, hrCNE variant 1 (hrCNE-1; Ref. 5) was used to separate the soluble proteins of the high molecular weight kit and the digitonin-solubilized mitochondrial membrane protein complexes from chicken heart (CH/Di) and from bovine heart mitochondria (BHM/Di). B, CNE could separate the proteins of the high molecular weight kit but failed to separate the digitonin-solubilized mitochondrial membrane protein complexes from chicken heart (CH/Di) sufficiently. Band M, monomeric ATP synthase. Band X was identified as a mixture of complex I and other respiratory complexes. C, mass calibration on hrCNE gel by soluble proteins of the high molecular weight kit (red triangles) and by digitonin-solubilized proteins from bovine heart mitochondria (black dots) and from chicken heart (green circles). D, mass calibration on a CNE gel by soluble proteins of the high molecular weight kit (red triangles). Mass/migration data were taken from supplemental Table S5. CAT, catalase; FER, ferritin; LDH, lactate dehydrogenase; TG, thyroglobulin.