6/2/2015BN-PAGE1 Analysis of membrane protein complexes by Blue native PAGE Veronika Reisinger and Lutz A. Eichacker Department for Biology I, Menzingerstr. 67, München

6/2/2015BN-PAGE2 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE3 Membrane isolation for BN-PAGE Isolate the membrane fraction. After isolation, pellet your membrane fraction and discard the supernatant. Do not use any denaturing or charged detergents, reductants or urea. Resuspend the membrane pellet in the buffer of your choice and determine the protein content: For membrane proteins we recommend Lowry et al., (1951) J. Biol. Chem. 193, Validation of your results. Please load per lane µg of protein (according to your protein determination) on a SDS-gel (mini-format, vertical unit electrophoresis system). We use 50 µg of total membrane protein per lane to yield protein bands well stainable with Coomassie. Identify the lane in which your proteins of interest are separated best and stained well. Multiply the amount of protein loaded onto this lane by the factor 8. This value corresponds to the amount of total membrane protein required for a standard BN-PAGE. Discard the supernatant and proceed with solubilization of your membrane fraction.

6/2/2015BN-PAGE4 Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE Workflow

6/2/2015BN-PAGE5 Detergents

6/2/2015BN-PAGE6 C 24 H 46 O 11, Mr = g/mol Dodecyl-ß-D-maltoside

6/2/2015BN-PAGE7 C 56 H 92 O 29, Mr = g/mol Digitonin glu gal xyl

6/2/2015BN-PAGE8 Comparison of Digitonin and ß-DM Digitonin (mmol/l) ß-DM (mmol/l) kDa AB

6/2/2015BN-PAGE9 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE10 Pre-PAGE labelling of proteins sensitivity limit quanti- fication in living cells dynamic range 3. Fluorescent dye, (Cy2, Cy3, Cy5) 100 pg yes 10 4 X-ray film P-imager plates 1 pg 0.2 pg +++ yes ++++ yes Stable isotope, ( 14 N / 15 N, 12 C / 13 C) < 1 pg ++++ (with MS) yes ? Detection limits: 1.Radio-isotopes, ( 35 S and 32 P),

6/2/2015BN-PAGE11 CyDye labeling of lysine side chains in native membrane protein complexes Cy5 633 nm Cy3 532 nm Cy2 EmEx 488 nm

6/2/2015BN-PAGE12 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE13 Casting a gradient gel from the top linear gradientexponential gradient

6/2/2015BN-PAGE14 Casting a gradient gel from the bottom Courtesy of HP Braun, University Hannover

6/2/2015BN-PAGE15 Electrophoretic parameters Size - mass - volume - structure Charge - pK-value - hydrate shell - Ion shell Buffer - pH-value - ionic strength - conductivity/resistance - viscosity - temperature Mobility - migration velocity Electric field - field strength (V/cm)

6/2/2015BN-PAGE16 Gel formation and pore size AGAROSEPOLYACRYLAMIDE Gelationofthepolysaccharidesolbychilling Chemicalpolymerisationofacrylamidemonomersand NN´-methylenbisacrylamide(Bis) 1%agarose(w/v)ca.150nm; 0.16%agarose(w/v)ca.500nm.    TotalacrylamideconcentrationandCrosslinking: T=100[%];C=100[%] a+b a+b a:gacrylamide;b:gBis;V:volumeinmL 5%T/3%C5nm   V b

6/2/2015BN-PAGE17 Velocity parameters v = E. µ = E. {z. e / (6   r)} Electrical field strengthE Voltage / Distance Mobilityµ [cm 2 / V x s] Suface charge of molecule( z. e) pH-dependent Size and form of molecule r Stokes Radius Viscosity of the medium h Buffermedium The velocity of an ion in an electric field is proportional to - field strength - charge inversly proportional to - size - viscosity of medium

6/2/2015BN-PAGE18 Masking the protein charges SDS, for denaturing SDS-PAGE Coomassie G250, for native protein complexes

6/2/2015BN-PAGE19 Charge state and separation of proteins by 2D Native/SDS-PAGE. 1.Dimension with LDS and Coomassie kDa 1.Dimension with ß-DM 2.Dimension (SDS-PAGE) 1.Dimension with ß-DM and Coomassie 2.Dimension (SDS-PAGE) ABC

6/2/2015BN-PAGE20 Molecular mass separation of protein complexes 1. Dimension (BN-PAGE) HMW (kD)  Protein-Complexes Distance/mass

6/2/2015BN-PAGE21 Sample application

6/2/2015BN-PAGE22 Running the first dimension BN-PAGE

6/2/2015BN-PAGE23 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE24 Denaturation of BN-PAGE strip and loading onto second dimension gel BN gel-lane Stacking gel Separating SDS gel SDS size marker Agarose

6/2/2015BN-PAGE25 Connecting 1st and 2nd dimension

6/2/2015BN-PAGE26 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE27 2. Dimension (SDS-PAGE) Principle of 2D BN-/SDS-electrophoresis PST (kD) Proteins 1. Dimension (BN-PAGE) kDa 2.Dimension (SDS-PAGE) 1. Dimension (BN-PAGE)

6/2/2015BN-PAGE28 Running second dimension SDS-PAGE

6/2/2015BN-PAGE29 Workflow Membrane isolation Solubilization of protein complexes BN-PAGE Cell fractionation Pre-PAGE labeling Post-PAGE analysis Denaturation of protein complexes SDS-PAGE

6/2/2015BN-PAGE30 sensitivity limit quanti- fication in living cells dynamic range 4. Coomassie 3. Negative 1. Silver 2. Fluorescent dye 100 ng 15 ng 200 pg 400 pg +++ no + ++ no ++++ no Staining: Post-PAGE staining of proteins

6/2/2015BN-PAGE31 Thanks to Reiner Westermeier, slides 10, 13, 16, 25, and 30 Bernd Müller, slides 20 and 27 HP Braun, slide 14 For contributing material used to generate these slides