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Procedures


Protein purification
Protein production in E. coli
Inclusion bodies
Electrophoresis
Chromatography
Concentrating proteins
Concentration determination
Protein chemical modifications
Protein chemical analysis

Protein crystallization
Lysozyme
Trypsin
Proteinase K

Protein X-ray crystallography

Protein structural analysis
Primary structure analysis
Secondary structure analysis
Tertiary structure analysis
Quaternary structure analysis

Structural bioinformatics

Publication

Protein purification




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Expression of recombinant genes in E. coli

LB media

1 L LB in 2L Erlenmeyer flask
10 g Tryptone
5 g Yeast extract
5 g NaCl
1 L DI water
Autoclave

100 mL LB in milk bottle
1.0 g Tryptone
0.5 g Yeast extract
0.5 g NaCl
100 mL DI water
Autoclave

100 mL LB-agar in milk bottle
1.0 g Tryptone
0.5 g Yeast extract
0.5 g NaCl
1.5 g Agar
100 mL DI water
Autoclave

Antibiotics

Ampicillin (stock 100 mg/mL)
1.0 g into 10 mL of DI water
Filter through 0.22-micron sterile filter into sterile 15 mL falcon tube
Aliquot into 1 mL volumes in sterile 1.5 mL microcentrifuge tubes and store in -20 C freezer

Ampicillin (working concentration 100 ug/mL)
Use 1 mL of stock in 1 L of media
Use 100 uL of stock in 100 mL of media

Kanamycin (stock 50 mg/mL)
0.5 g into 10 mL of DI water
Filter through 0.22-micron sterile filter into sterile 15 mL falcon tube
Aliquot into 1 mL volumes in sterile 1.5 mL microcentrifuge tubes and store in -20 C freezer

Kanamycin (working concentration 50 ug/mL)
Use 1 mL of stock in 1 L of media
Use 100 uL of stock in 100 mL of media


IPTG: Isopropyl-beta-D thiogalatopyranoside (238.30 g/mol)

IPTG (stock 1 M )
2.38 g into 10 mL of DI water
Filter through 0.22-micron sterile filter into sterile 15 mL falcon tube
Aliquot into 1 mL volumes in sterile 1.5 mL microcentrifuge tubes and store in -20 C freezer

IPTG (Working concentration 0.5 mM)
Use 0.5 mL of stock in 1 L of culture (optical density = 0.6-0.8)


Glycerol Stocks

50% glycerol in a milk bottle (PYREXâ„¢ Milk Dilution Bottles with Screw Cap)
50 mL DI water
50 mL glycerol
mix
Autoclave
Let cool

Making glycerol stock
Prepare overnight cell culture with antibiotic In screw cap 2 mL vials add:
500 mL overnight cell culture
500 mL of 50% glycerol
Gently mix fully
Put into -80 degree C freezer
Label vial with sharpie before putting into freezer

Isolating, washing, solubilizing and refolding inclusion bodies

Lysis using sonication

Thaw cells (in 50 mL conical tube)
Suspend cells, in 10 mL of lysis buffer per gram of cells
Wear ear protection
Clean probe with alcohol
Press No microtip
Press Operate manually
Up arrow to Amplitude of 20
1 min. at 20 power
1 min. rest on ice
1 min. at 30 power
1 min. rest on ice
1 min. at 40 power
1 min. rest on ice
1 min. at 40 power
1 min. rest on ice
1 min. at 40 power
Clean probe with alcohol

Separating inclusion bodies

Use 40 mL centrifuge tubes
Use JA 25.50 rotor
5,000 rpm for 15 min.
Pour off supernatant, save in a separate tube
Suspend inclusion bodies in 20 mL of inclusion bodies wash buffer
Use a small stir bar on a stir plate (use beaker to hold up tube)
Stir for ~ 20 min.
Take 20 uL sample for SDS-PAGE gel analysis
Remove stir bar
Centrifuge
Repeat

Lysis buffer

20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA (1L)
2.42 g of Tris (121.14 g/mol)
5.84 g of NaCl (58.44 g/mol)
0.29 g of EDTA (292.24 g/mol)
Add ~975 mL DI water
Stir
cool to 4 degree C overnight
pH to 8.0
QS to 1000 mL

Inclusion body wash buffer

20 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 % Triton X-100 (1L)
2.42 g of Tris (121.14 g/mol)
2.92 g of EDTA (292.24 g/mol)
5 mL of Triton X-100
~975 mL DI water
Stir
cool to 4 degree C overnight
pH to 8.0
QS to 1000 mL

Guanidinium chloride

4 M Guanidinium chloride (1 L)
381.2 g Guanidinium chloride (95.53 g/mol)
~ 500 mL DI water
Stir (endothermic reaction)
QS to 1 L with DI water
Store at 4 °C

Dilution buffer

20 mM Tris-HCl pH 8.0, 0.5 % Triton X-100 (1L)
2.42 g of Tris (121.14 g/mol)
5 mL of Triton X-100
~975 mL DI water
Stir
cool to 4 degree C overnight
pH to 8.0
QS to 1000 mL

Dialysis buffer

20 mM Tris-HCl pH 8.0
~975 mL DI water
Stir
cool to 4 degree C overnight
pH to 8.0
QS to 1000 mL


SDS-PAGE

SDS-PAGE Reagents

10% SDS (100 mL)
10 g Sodium dodecyl sulfate (SDS)
~ 90 mL DI water
Stir gently with stir bar
QS to 100 mL with DI water
Store at room temperature

30% Acrylamide (300 mL) (500 mL beaker)
87.6 g acrylamide (Caution neurotoxin when in solution!)
2.4 g N’N’ bis methylene acrylamide (Caution neurotoxin when in solution!)
~ 250 mL DI water and stir with stir bar (takes some time to dissolve – endothermic)
QS to 300 mL in 500 mL graduated cylinder
Store at 4 degree C in dark

1.5 M Tris-HCl pH 8.8 (150 mL)
27.2 g Tris-base
Add 80 mL DI water
pH to 8.8 with HCl
QS to 150 mL
Store at 4 degree C

0.5 M Tris-HCl pH 6.8
6.0 g Tris-base
~60 mL DI water
pH to 6.8 with HCl
QS to 100 mL
Store at 4 degree C

Coomassie Stain (250 mL) BioRad instructions
40% Methanol (100 mL)
10% Acetic acid (25 mL)
50% water (125 mL)
0.250 g R-250

5x running buffer pH 8.3
9.0 g Tris-base
43.2 g Glycine
3.0 g SDS
600 mL DI water

1x running buffer
90 mL 5X + 360 mL DI water

10% Ammonium Persulfate solution (APS)
0.10 g ammonium persulfate into 1.5 mL microcentrifuge tube
Add 1 mL of DI water

N, N, N', N'-tetramethylethylenediamine (TEMED)
Make sure it is a fresh bottle (not older than a yr since purchase)

2X sample buffer (BioRad formulation, 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% BME)
Into a 50 mL falcon tube add:
3.75 mL 0.5 M Tris pH 6.8
15 mL 50% Glycerol
0.3 mL 1.0% bromophenol blue
6.0 mL 10% SDS
4.95 mL DI water
Vortex
Aliquot into 0.95 mL volumes (1.5 mL microcentrifuge tubes)
Store at room temperature

To use: vortex and then add 50 uL beta-mercaptoethanol (BME, or 2-mercaptoethanol) and vortex before use

1% bromophenol blue (w/v)
Add 0.100 g bromophenol blue in 15 mL falcon tube
Add 10 mL DI water
Stir on vortex

Overlay solution
water-saturated n-butanol (butanol is the top layer) in glass bottle
50 mL n-butanol
5 mL DI water
Shake
Use top layer only, store at RT
Apply with a Pasteur pipette and bulb

Preparing gels:
Equilibrate solutions to RT (23–25 degree C) before polymerization
Let gel polymerize for at least 2 hr
Prepare fresh APS solution (daily)
Replace TEMED every three months
Clean glass plates with ethanol and kimwipe

15% Separating gel
2.4 mL water
5.0 mL 30% acrylamide
2.5 mL 1.5 M Tris pH 8.8
100 uL SDS

100 uL APS
10 uL TEMED

4% Stacking gel
6.1 mL water
1.33 mL 30% acrylamide
2.5 mL 0.5 M Tris pH 6.8
100 uL SDS

100 uL APS
10 uL TEMED

Preparing samples
10 uL protein + 10 uL 2X sample buffer
Heat at 95 degree C (in heating block) for 4 minutes*
Let cool for 4 minutes
Quick spin to bring down vapor
*Note: Sometimes it is best to not heat membrane proteins

Running Electrophoresis
200V, ~ 40 min
Do not reuse running buffer






Protein Crystallization


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Lysozyme crystallization

NaCl conditions

Protein:
Sigma #L7651, 3 times crystallized chicken (Hen) egg white lysozyme - HEWL ( (20 mg/mL) in 20 mM sodium acetate trihydrate pH 4.6
Centrifuge in microcentrifuge
Filter through 0.22-micron sterile filter

Well:
a) 0.6 to 1.5 M Sodium chloride, 0.1 M Sodium acetate trihydrate pH 4.0 to 4.8
b) 0.6 M Sodium chloride, 0.1 M sodium acetate trihydrate pH 4.2 to 4.8, 25% ethylene glycol
c) 0.6 M Sodium chloride, 0.1 M sodium acetate trihydrate pH 4.2 to 4.8, 25% glycerol
d) 30% w/v Polyethylene glycol monomethyl ether 5,000, 1.0 M sodium chloride, 50 mM sodium acetate trihydrate pH 4.5
e) 5% w/v Sodium chloride, 0.1 M sodium acetate trihydrate pH 4.2 to 4.8
f) 10 % w/v Sodium chloride, 5% v/v propanol
g) 0.44 M Sodium nitrate, 100 mM sodium acetate pH 4.6

Drop:
3 uL protein solution and 3 uL of well solution

Space Group and typical Unit Cell:

Sodium Chloride / Sodium Acetate Condition
P43212
a,b,c (Angstroms) = 78.8, 78.8, 37.0
α,β,γ (degrees) = 90.0, 90.0, 90.0

Sodium Nitrate / Sodium Acetate Condition
P1
a,b,c (Angstroms) = 26.8, 31.1, 33.8
α,β,γ (degrees) = 89.2, 72.5, 67.9


Trypsin crystallization

Condition 1
Protein:
Sigma #T8253, Bovine Pancreas Trypsin (60 mg/ml) in 10 mg/ml benzamidine, 3 mM calcium chloride, and 0.02% (w/v) sodium azide
Centrifuge in microcentrifuge
Filter through 0.22-micron sterile filter

Well:
4%-8% (w/v) PEG4000, 0.2 M Li2SO4, 0.1 M MES pH 6.5, and 15% ethylene glycol

Drop:
3 uL protein solution and 3 uL of well solution


Proteinase K crystallization

Condition 1
Protein:
Sigma #P2308 (#539480/ CAS#: 39450-01-6), Tritirachium album Proteinase K. A subtilisin like fungal serine protease (Asp39-His69-Ser224). Suspend in 50 mM HEPES pH 7.0 (10-20 mg/ml)
Centrifuge in microcentrifuge
Filter through 0.22-micron sterile filter

Well:
Condition 1: 1.2 M Ammonium Sulfate, 0.1 M Tris-HCl pH 8.0
Condition 2: 1.0 M NaNO3, 100 mM Citrate Buffer pH 6.5
Drop:
3 uL protein solution and 3 uL of well solution



Protein X-ray crystallography

:The Canadian Synchrotron Light Source (CLS)
:Structural Biology Synchrotron Users Organization
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Protein structural analysis


Primary structure analysis

Servers










Sequence alignment figure with secondary structure and annotations
1. Collect FASTA format sequences from UniProt
2. Perform multiple alignment with Clustal Omega, save as a text file (seq.aln)
3. ESPript 3.0
Use ADV
Upload pdb
choose chain
To put a black star at catalytic residues:
under special commands and characters
S D ##
S D ###
Submit


Secondary structure analysis

Servers




Tertiary structure analysis

PyMol





PyMol scripts

General settings for making figures:

hide everything;
set valence, 0;
bg_color white;
set ray_shadows, 0;
set fog, off;


Color:

Color any selection (atom/chain etc.) to any color

color color-name, (selection);

Bauhaus colors

color blue, (chA);
color red, (chB);
color yellow, (chC);


Color spectrum N(blue) to C(red).

spectrum count, rainbow, chain A, byres=1;


Color by B-factor

spectrum b, selection=chA;


Color by Element

color atomic, (not elem C);


Ball&Stick:

set stick_ball, on;
set stick_ball_ratio, 1.5;
set stick_radius, 0.17000;


Scenes:
Helpful for presentations and in saving your work during protein structural analysis
Button to start scenes at top menu
Can rename scenes

Lines:
Use measure to make dashed line between atoms
Edit all: dash

Hydrogen Bond Lines:

set dash_color, black;
set dash_gap, 0.35;
set dash_length, 0.10;
set dash_width, 4.5;


To Show Hydrogens:
set stick_h_scale, 1.0;

Labels:
to position the position of labels

set label_position,(2,2,3);


Selections:

Selection specific residues and ranges of residues:

select binding_site, (resi ##-##+##+##+###-### and chain A);>


Selection residues within chain A that are within 5 Angstroms of chain B:

select chA_contact, chain A and byres all within 5 of chain B;


Selection residues within chain B that are within 5 Angstroms of chain A:

select chB_contact, chain B and byres all within 5 of chain A;


Selection specific residue types:

select aas, resn asp+glu+asn+gln;


Select alpha-carbons:

select ca, name ca #;


Select a region of residues:

select active, (resi 14-20, 38 and chainA);


Select all residues within a defined distance from a ligand or a residue or an atom:

select active, byres all within 5 of ligand;


To select all waters within a defined distance from a ligand:

select ligand_water, ((ligands) around 3.2) and (resn HOH);


To select all waters within a defined distance from an active site:

select active_water, ((active) around 3.2) and (resn HOH);


alter active_water, vdw=0.5;
rebuild;

or

alter Mg, vdw=1;
rebuild;


Structural Alignments:

align mobile, target;

(for proteins with >30% ID)

super mobile, target;

(for proteins with <30% ID)

super mobile & chain, target & chain;

(useful when you have >1 chain in the objects you want to align, aligning one chain & bringing along the other chain(s))

cealign target, mobile;

(for proteins with <30% ID)

Notes: The molecules you want to align need to be in two different objects

Calculating solvent accessible surface area (SASA):
Notes: Load 3 separate molecules: protein1, protein2, protein1_protein2_complex

Show dot;
set dot_solvent, on;
set dot_density, 4;
get_area protein1;
get_area protein2;
get_area protein1_protein2_Complex;

Notes: Buried surface = (protein1_area + protein2_area) - protein1_protein2_complex_area

Surfaces:
Semitransparent surface

bg_color white;
set ray_shadows,0;
set surface_quality, 1;
set transparency, 0.4;


Rendering Surfaces:
For a black silhouette

set surface_color, black;
set transparency, 0;
set ray_trace_mode,3;


For a grey silhouette

set surface_color,black;
set transparency, 0.5;
set ray_trace_mode,3;


For a surface with black outline

set surface_color, black
set transparency, 0;
set ray_trace_mode,2;


For a surface with black/white alternating

set surface_color to black/White alternating
set transparency, 0;
set ray_trace_mode,2;

turn off everything under Display (Specular Reflection etc…)

Ray Trace Mode:
These modes are best run with antialiasing set to 1 to 3

set antialias, 2;
set bg_color, white;


# normal color

set ray_trace_mode, 0;


# normal color + black outline

set ray_trace_mode, 1;


# black outline only (B/W outline)

set ray_trace_mode, 2;


# quantized color + black outline

set ray_trace_mode, 3;


Surface analysis




Binding site / active site analysis




Other sites





Quaternary structure analysis

Servers




Structural bioinformatics







Publication

Journals






















































Review Journals











Scientific Writing