04/21/23 BN-PAGE 1

Analysis of membrane protein complexes by Blue native PAGE

Veronika Reisinger and Lutz A. EichackerDepartment for Biology I, Menzingerstr. 67, 80638 München

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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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, 265-

275.

• Validation of your results. Please load per lane 1-100 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.

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Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

Workflow

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Detergents

Name Description MW monomer

Aggregation number

MW micelle

CMC %(w/v)/(mM)

Na-Deoxycholate Anionic 415 4-10 700-9000 0.11/(1-2.7)

SDS Anionic 288 62 18000 0.24/(8.2)

Taurodeoxycholate Anionic 522 4 4200-30000

0.05-0.2/(1-4)

Digitonin Nonionic 1229 5-6 70000 0.031/(0.25)

Dodecylmaltoside Nonionic 511 98 50000 0.008/(0.16)

Octylglucoside Nonionic 292 27 8000 0.73/(25)

Triton-X-100 Nonionic 650 140 90000 0.02/(0.24)

Tween 20 Nonionic 1228 - - 0.0074/(0.06)

Chaps Zwitterionic 615 10 6150 0.25/(4)

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C24H46O11 , Mr = 510.62 g/mol

Dodecyl-ß-D-maltoside

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C56H92O29, Mr = 1229.31 g/mol

Digitonin

glu

gal

xyl

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Comparison of Digitonin and ß-DM

669

440

232

140

67

669

440

232

140

67

2.21.1 4.5 9.0 18.0 36.0 72.0

Digitonin (mmol/l)

2.21.1 4.5 9.0 18.0 36.0 72.0

ß-DM (mmol/l)

kDa kDa

A B

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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Pre-PAGE labelling of proteins

sensitivity limit

quanti-fication

in living cells

dynamicrange

3. Fluorescent dye, (Cy2, Cy3, Cy5) 100 pg ++++++ yes104

X-ray film

P-imager plates1 pg

0.2 pg

+++yes

++++yes

20

105

2. Stable isotope, (14N /15N, 12C /13 C) < 1 pg ++++ (with MS)

yes?

Detection limits:

1. Radio-isotopes, (35S and 32P),

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CyDye labeling of lysine side chains in

native membrane protein complexes

Cy5633 nm

Cy3532 nm

Cy2EmEx488 nm

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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Casting a gradient gel from the top

linear gradient exponential gradient

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Casting a gradient gel from the bottom

Courtesy of HP Braun, University Hannover

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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)

+

+

+

++

+

+

+

+

+

+

+

-

+

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Gel formation and pore size

AGAROSE POLYACRYLAMIDEGelation of the polysaccharide sol by chilling Chemical polymerisation of acrylamide monomers and

NN´-methylenbisacrylamide (Bis)

1% agarose (w/v) ca. 150 nm;0.16 % agarose (w/v) ca. 500 nm.

Total acrylamide concentrationand Crosslinking:

T = 100 [%]; C = 100 [%]a + ba + b

a:g acrylamide; b:g Bis;V: volume in mL

5 % T / 3 % C 5 nm

V

b

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Velocity parameters

v = E . µ = E . {z . e / (6 r)}

• Electrical field strength E Voltage / Distance• Mobility µ [cm2 / V x s]• Suface charge of molecule (z . e) pH-dependent• Size and form of molecule r Stokes Radius• Viscosity of the medium Buffermedium

The velocity of an ion in an electric field is

proportional to - field strength - charge

inversly proportional to

- size- viscosity of medium

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Masking the protein charges

SDS, for denaturing SDS-PAGE

Coomassie G250, for native protein complexes

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Charge state and separation of proteins by 2D Native/SDS-PAGE.

1.Dimension with LDS and Coomassie

669 440 232 140 67669 440 232 140 67kDa kDa

1.Dimension with ß-DM

2.D

imen

sion

(S

DS

-PA

GE)

1.Dimension with ß-DM and Coomassie

2.D

imensi

on

(SD

S-P

AG

E)

2.D

imensi

on

(S

DS

-PA

GE)

A B C

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Molecular mass separation of protein complexes

1. Dimension (BN-PAGE)

HMW (kD)

669

440

232

66

140

669

Protein-Complexes

y = 1008.6e -3.1133x

R2 = 0.9893

10

100

1000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1rf-value

kDa

molecular mass standard proteins

cpHSP60

RCII Core(2)

RubisCo

ATPase

LHCII(3)

POR(2)

POR(1)

RCII Core(1)

Cyt b6/f

Distance/mass

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Sample application

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Running the first dimension BN-PAGE

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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Denaturation of BN-PAGE strip and loading onto second dimension gel

BN gel-lane

Stacking gel

Separating

SDS gel

SDS size marker

Agarose

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Connecting 1st and 2nd dimension

1 mm thin cassette spacers

Method 1

1,5 mmthick BN gel

acrylamide monomer solution

Method 2

1 mm thin cassette spacers

0,75 mm thin BN gel

polyacrylamidegel

agarose

acrylamide monomer solution

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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2.

Dim

ensi

on (

SD

S-P

AG

E)

Principle of 2D BN-/SDS-electrophoresis

199

133

87

40.1

31.6

18.5

7.1

PST (kD)

Proteins

1. Dimension (BN-PAGE)

669 440 232 140 67kDa

2.D

imensi

on (

SD

S-P

AG

E)

1. Dimension (BN-PAGE)

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Running second dimension SDS-PAGE

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Workflow

Membrane isolation

Solubilization of protein complexes

BN-PAGE

Cell fractionation

Pre-PAGE labeling

Post-PAGE analysis

Denaturation of protein complexes

SDS-PAGE

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sensitivity limit

quanti-fication

in living cells

dynamicrange

4. Coomassie3. Negative

1. Silver

2. Fluorescent dye

100 ng15 ng

200 pg

400 pg

+++no+no

++no

++++no

33

7

104

Staining:

Post-PAGE staining of proteins

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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