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Molecular Biotechnology
 
October 1 - 12
Paula Veríssimo, Luísa Cortes, Bruno Manadas, Mónica Sousa
Lab. Biotecnologia Molecular do CNC
Dep. Bioquímica, Universidade de Coimbra
IBMC


Importante:
A aula de 1 de Outubro será dada no Anfiteatro de Fisiologia, 1º piso
Course Schedule

 
Monday
1 Oct.
Tuesday
2 Oct.
Monday
8 Oct
Tuesday
9 Oct.
Wednesday
10 Oct.
Thursday
11 Oct.
Friday
12 Oct.
9-10
Lect.1
Lect. 5
Lect. 9
Lab.
Lab.
Lab.
 
10-11
Lect.2
Lect.6
 
Lab.
Lab.
Lab.
Lect. 10
11-12
 
 
 
Lab.
Lab.
Lab.
 
12-13
 
 
 
Lab.
Lab.
Lab.
Overview of
 
 
 
 
 
 
 
Pratical sessions
14-15
Lect. 3
Lect. 7
Lab.
Lab.
Lab.
Lab.
 
15-16
Lect. 4
Lect. 8
Lab.
Lab.
Lab.
Lab.
 
16-17
 
 
Lab.
Lab.
Lab.
Lab.
 
17-18
 
 
Lab.
Lab.
Lab.
Lab.
 

Lectures
  • Lect.1: Cloning strategies
  • Lect. 2& 3: Expression of cloned genes in prokaryotes
  • Lect. 3&4: Expression of cloned genes in eukaryotes   
  • Lect. 5: Recombinant protein production
  • Lect. 6&7: Protein purification
  • Lect. 8: Protein engineering
  • Lect. 9: Proteomics
  • Lect. 10: Protein aggregation and disease
Pratical course
  • Purification and characterisation of native proteins.
  • Expression of fusion proteins.
  • Expression, purification and characterisation of recombinant proteins.
Reading Lists
Purification and characterization of cardosins: aspartic proteinases from Cynara cardunculus L.
  • Homogenization of pistils from Cynara cardunculus with extraction buffer
  • Centrifugation of total extract
  • Purification of cardosins
  • Evaluation of the purification process by SDS-PAGE and Western blot
  • Enzymatic characterization
Expression of recombinant PSI (Plant-specific Insert
  • Growth of E. coli BL21 cells transformed with recombinant plasmid
    pGST-PSI
  • Induction of protein expression by IPTG
  • Harvest of cell pellet by centrifugation
  • Purification of GST-PSI by affinity chromatography with glutathione-Sepharose.
  • Analysis of the purification process by SDS-PAGE and Western blot
Expression, purification and characterization of recombinant cardosin A 
  • Growth of E. coli BL21 cells transformed with recombinant plasmid
    pET-pCA
  • Induction of protein expression with IPTG
  • Harvest of cell pellet by centrifugation
  • Isolation of inclusion bodies
  • Protein refolding
  • Purification of recombinant cardosin A
  • Evaluation of the purification process by SDS-PAGE
  • Enzymatic characterisation
Miscellaneous Bytes of Information
http://www.embl-heidelberg.de/srs5/            Sequence Retrieval System: SRS5 server
http://www.expasy.ch/srs5/
http://www.expasy.ch/cgi-bin/sprot-search-de         Search of sequences in SwissProt and TrEMBL
http://www.ncbi.nlm.nih.gov/Entrez/protein.html   Search of protein sequences in NCBI database
http://bmbsgill.leeds.ac.uk/bmb5dp/owl.html           Address of OWL
http://www.expasy.ch/ch2d/2d-index.html               2D databases
http://www.mdc-berlin.de/~emu/heart/                   2D Human Heart database
http://www.rcsb.org/pdb                                        3D database
http://vega.crbm.cnrs-mop.fr/bin/fasta-guess.cgi    Genestream FastA
http://www2.ebi.ac.uk/fasta3/                                FastA
http://www.expasy.ch/cgi-bin/BLASTEMBnet-CH.pl     BLAST
http://www.ncbi.nlm.nih.gov/blast/blast.cgi               BLAST
http://bmbsgill.leeds.ac.uk/bmb5dp/blast/blast.html BLAST search of OWL
http://www.expasy.org/tools                                     ExPASy Proteomics tools Last http://www2.ebi.ac.uk                      Database Searching, Browsing, and Analysis Tools
http://www.ebi.ac.uk /msd                Tools for PDB analysis                                
http://www.fmi.ch/biology/research_tools.html    Pedro`s BioMolecular Research Tools
http://prowl.rockefeller.edu/    PROWL-for protein chemistry and mass spectrometry
http://www.proteometrics.com  Protein info, search with keywords or sequences
http://www.embl-heidelberg.de/predictprotein/predictprotein.html   Prediction of secondary structure
http://www.nwfsc.noaa.gov/protocols/                  Molecular Biology Protocols
http://www.nwfsc.noaa.gov/protocols/bioinformatics.html / Molecular Biology Protocols
http://www.bioinforman.ac.uk/dbbrowser/bioactivity/           Bioinformatic Practical          
 
Protocols
Expressão de cardosina A recombinante em E.coli
Protocolo para 1litro de meio LB
1. Repicar colónia da placa ZB para 50 ml ZB ao qual se adicionou 15 µl de ampicilina ( stock: 100 mg/ml )
2. Incubar ON a 30 °C, 155 rpm
3. Passar cerca de 15 ml da incubação O.N para LB/ampicilina (150 ml de ampicilina do stock 100 mg/ml  por cada ½ Litro L.B)
4. Crescer a 37 °C, 180 rpm durante cerca de 2 – 2:30h até se atingir uma DO600  ~ 0.6-0.7
5. Adicionar IPTG para uma concentração final de 0.119 g/L (concentração final 0.5mM)
6. Continuar a incubação durante 3 horas a 37 °C, 180 rpm
7. Centrifugar 6.000 rpm, 15 min.
8. Ressuspender o sedimento em tampão TN (~ 50 ml )
9. Adicionar um pouco de lisozima (na ponta da espátula)
10. Congelar o sedimento assim ressuspenso a – 20 °C durante a noite
11. Descongelar à temperatura ambiente, agitando ocasionalmente de modo “ gentil “
12. Após descongelar, adicionar 1ml MgCl2 (1M) e agitar suavemente com a espátula
13. Adicionar DNAase I
14. Agitar suavemente e incubar em gelo com agitação ocasional até se observar uma consistência bastante líquida
15. Adicionar o conteúdo a 1 litro de tampão TN e manter a agitar a 4 °C pelo menos durante 2 horas
16. Centrifugar 6.500 rpm, 20 min., 4 °C
17. Ressuspender o pellet em 1 L de tampão TNT
18. Manter a 4 °C com agitação pelo menos durante 2 horas (pode ser on)
19. Centrifugar 6.500 g, 20 min., 4 °C
20. Ressuspender em ~ 40 ml de tampão 8M Ureia
21. Adicionar β – mercaptoetanol (7 ml/litro)
22. Incubar sob agitação a 4 °C até se obter uma solução homogénea
 
Tampões:

LB:     10gr NaCl
10gr Triptona            1 Litro, pH 7.5 ( aprox.)
5grextracto de
 levedura
ZB:      10gr N-Z-amina
5gr NaCl        1litro  
TN (20 ×): 1 M Tris   pH 7.4 1×: Diluir TN (20 X) 50 ml em 950ml H2O                            1 M NaCl
TNT: TN (1 X), ( 990 ml ) + 10 ml Triton ×-100
 
8 M Ureia: 8 M Ureia
            0.1M Tris                   pH 10.5
1 mM glicina
1 mM EDTA
 
Purificação da proteína recombinante
1. Cromatrografia de exclusão molecular ( baixa pressão ) Sephacryl S-300, Tampão: 20 mM Tris, 0,4M Ureia pH 8.0 Fluxo: ~ 0.5 ml/min
Aplicam-se até 10 ml da proteína numa coluna com aprox. 100 × 26 mm.
2. Cromatografias de troca iónica :
Econo High Q (5 ml)
Mono Q (1ml)
    Tampão A: Tris 20 mM,0,4M Ureia pH 8.0
    Tampão B: Tris 20mM, 0,4M Ureia pH 8.0, 1 M NaCl
 
Expressão de GST-PSI em E.col
1. Repicar colónia da placa LB para 20 ml LB ao qual se adicionou 10 µl de ampicilina ( stock: 100 mg/ml )
2. Incubar ON a 30 °C, 155 rpm
3. Passar cerca de 10 ml da incubação O.N para 500ml de LB/ampicilina (250 ml de ampicilina do stock 100 mg/ml )
4. Crescer a 37 °C, 180 rpm durante cerca de 3h até se atingir uma DO600  ~ 0.6-0.7
5. Adicionar IPTG para uma concentração final de 0.119 g/L (concentração final 0.5mM)
6. Continuar a incubação durante 3 horas a 37 °C, 180 rpm
7. Centrifugar 6.000 rpm, 15 min.
8. Ressuspender o pellet celular em TBS 1x (~ 25 ml )
9. Adicionar um pouco de lisozima (na ponta da espátula)
10. Congelar o sedimento assim ressuspenso a – 20 °C durante a noite
11. Descongelar à temperatura ambiente, agitando ocasionalmente de modo “ gentil “
12. Após descongelar, adicionar 1ml MgCl2 (1M) e agitar suavemente com a espátula
13. Adicionar DNAase I
14. Agitar suavemente e incubar em gelo com agitação ocasional até se observar uma consistência bastante líquida
15.Centrifugar 12000 rpm, 10 min., 4 °C e guardar o sobrenadante para incubação com a resina.~25ml.
Purificação da proteína de fusão
Preparação da resina Glutationa –Sepharose:
  • Retirar 800ul de suspensão de resina e lavar com TBS ~10 volumes de coluna.
  • Incubar o sobrenadante com a resina 1hora a 4º C com agitação.
  • Empacar a coluna 1ml
  • Lavar com TBS 10 vol. (recolha do 1ª e ultimo vol.)
  • Eluição é feita com glutationa reduzida. ~400ul de glutationa na coluna fechada incubando 8 a 10 min.
  • São realizadas 4 eluições
SDS/Polyacrylamide Gel Electrophoresis
Standard SDS-polyacrylamide gel electrophoresis (Laemmli)--gel preparation. Volumes given are sufficient for small (8 cm X 10 cm X 1.5 mm) gel format (10 ml of monomer). Scale up volumes as needed.
1. Pour the Separating Gel
Set up your gel apparatus, prepare separating gel monomer. Add TEMED just prior to pouring gel (I "pour" the gels using a pasteur pipet and a rubber bulb). Allow to polymerize before adding stacking gel by overlaying gently with water or n-butanol. With higher % gels, one can immediately pour the stacking gel on the unpolymerized separating gel. Be careful not to mix the two layers.
 

Separating Gels, in 0.375 M Tris, pH 8.8
 
 
 
 
 
 
7%
10%
12%
15%
distilled H2O
5.1 ml
4.1 ml
3.4 ml
2.4 ml
1.5 M Tris-HCl, pH 8.8
2.5 ml
2.5 ml
2.5 ml
2.5 ml
20% (w/v) SDS
0.05 ml
0.05 ml
0.05 ml
0.05 ml
Acrylamide/Bis-acrylamide
(30%/0.8% w/v)
2.3 ml
3.3 ml
4.0 ml
5.0 ml
10% (w/v) ammonium persulfate
0.05 ml
0.05 ml
0.05 ml
0.05 ml
TEMED
0.005 ml
0.005 ml
0.005 ml
0.005 ml
Total monomer
10.005 ml
10.005 ml
10.005 ml
10.005 ml

2. Pour the Stacking Gel
After the separating gel has polymerized, decant the overlay, prepare the stacking monomer, add the TEMED, and pour. Insert the comb and allow to polymerize completely before running.
 

Stacking Gels, 4.0% gel, 0.125 M Tris, pH 6.8
distilled H2O
3.075 ml
0.5 M Tris-HCl, pH 6.8
1.25 ml
20% (w/v) SDS
0.025 ml
Acrylamide/Bis-acrylamide
(30%/0.8% w/v)
0.67 ml
10% (w/v) ammonium persulfate
0.025 ml
TEMED
0.005 ml
Total Stack monomer
5.05 ml

For best results:
1. Make ammonium persulfate solution fresh daily.
2. Degas solutions before adding TEMED for 15 min at room temperature.
3. Running the gel
I usually run my gels at constant current, 25-50 mA, depending on gel size. Here´s the recipe for 5X SDS-PAGE running buffer. Dilute to 1X before use.
 

5X Running Buffer, pH 8.3 (1 liter)
Tris Base
15 g
Glycine
72 g
SDS
5 g
distilled water to 1 liter

Store at room temperature until use.
4. Sample buffer
Dilute samples at least 1:4 with sample buffer, heat at 95 C for 4 minutes prior to loading.

Sample Buffer (8 ml)
Distilled water
4.0 ml
0.5 M Tris-HCl
1.0 ml
Glycerol
0.8 ml
10% SDS
1.6 ml
beta-mercaptoethanol
0.4 ml
0.05% (w/v)
bromophenol blue
0.2 ml

 
Western Blot

1) Run protein gel. 

Set up blot:

2)
Prepare two tupperware containers, one with dH2O and one with 1/2X Transfer Buffer (TB).
3) Remove one glass plate from the gel by twisting a spacer between the plates. To remove the other plate, place the gel in the TB container and gently scrap off with a flat scoopula. Let the gel soak for about 5 mins.
4) To make the blotting sandwich, begin by putting the blotting apparatus in the TB container with the black side down. Then put on a pad and one piece of paper (make sure both are completely wet in the TB). Next the gel goes on.
5) Use the tweezers to grab the nitrocellulose membrane and dunk it in the dH2O container, make sure the entire membrane is wet, and then move it to the TB. When it is completely soaked with TB, move it onto the sandwich next.
6) To complete the sandwich, one more piece of paper goes on and then the last pad, again making sure both are thoroughly soaked in TB.
7) Roll a 15ml tube up and down and side to side over the completed sandwich to remove any bubbles. Close the blotting apparatus securely.
8) Put a small stir bar in the gel box underneath the black and red sleeve. Then put the ice pack in next to the sleeve.
9) Put the blotting sandwich into the black and red sleeve with the black part of the sandwich facing the black part of the sleeve. Then fill the gel box up to the top of the red half of the sleeve with TB.
10) Run the blot on manual and constant V at 100V for about 1 hour.

Staining the blot:

11)
Dump out the TB, and remove the sandwich.
12) Use tweezers to put membrane into a small tupperware top and cover with ponso (red stuff). Leave on the orbital shaker or rocker for about 15 mins.
13) Rinse with water until markings appear. Use a pencil to mark the ladder, and distinguish the 50kDa marker somehow. Cut the top right-handcorner so that "up" is easily recognizable.
14) Wash with block 10-15 mins. on the rocker.
15) Transfer to the smallest container possible.

Binding the antibody

16)
Add diluted primary antibody.
17) Incubate primary antibody overnight at 4 C.
18) Wash 3 times for 15 mins. each with 1XTBST on a rocker.
19) Dilute out secondary antibody 1: 5,000 (1µl in 5ml).
20) Dump off third wash and put in secondary antibody mix. Incubate 1 hr. on a rocker.
21) Wash 3 times for 15mins. each with 1XTBST on a rocker.

Developing the blot:

22)
Materials needed: Two pieces of saran-wrap, detection reagent 1 and 2 (bottles at 4 C with a big ECL on the label, one with a white top and one with a black top), film cassette, scissors, tweezers and film.
23) Mix 2ml of 1 and 2ml of 2 in a 15ml tube (BE SURE TO CHANGE PIPETS IN BETWEEN REAGENTS).
24) Use tweezers to move membrane on to a piece of saranwrap and drip the reagent mix all over the membrane. Let stand for 1min.
25) Drip off excess mix, and transfer the membrane to a clean piece of saranwrap upside down. Wrap the membrane up in the saranwrap and place it into the film cassette right-side up.
26) Bring the cassette, film and scissors in a darkroom with a developer. When in the darkroom, cut a piece of film to fit the cassette.
27) Burn the film for 1min, bend the top right hand corner down and back again to distinguish the correct orientation.
28) Slide the film through the developer to develop.
WESTERN BLOT:

Solutions:

a) 1.5 M Tris-HCl pH 8.8
Dissolve 54.5 g Tris base in 200 ml of distilled water.
Adjust pH to 8.8 using concentrated HCl. Make up total volume to 300 ml with distilled water. Store at 2-8 C.

b) 0.5 M Tris-HCl pH 6.8
Dissolve 12.1 g of Tris base in 150 ml of distilled water. Adjust pH to 6.8 using concentrated HCl Make up total volume to 200 ml with distilled water. Store at 2-8 C

c) 10% (w/v) Sodium dodecyl sulfate (SDS)
Dissolve 5 g of SDS in 50 ml of distilled water. Mix until dissolved. Store at room temperature.

d) 10% Bromphenol blue
Dissolve .1 g in 1 ml of distilled water. Store at room temperature.

e) SDS-PAGE loading buffer
In a 20 ml vial mix the following:

4.2 ml water
1.0 ml 0.5 M Tris-HCl pH 6.8
0.8 ml glycerol
1.6 ml 10 % (w/v) SDS
0.4 ml 2-mercaptoethanol
0.02 ml 10% (w/v) bromphenol blue

Store at room temperature

It is recommended that the above recipe is used because alternative tracking dyes or even an excess of dye will give fluorescent bands at the gel front, which may interfere with detection of the protein of interest.


f) 10% (w/v) Ammonium persulphate (AMPS)
Dissolve 0.1 g of AMPS in 1 ml of water. USE IMMEDIATELY.

g) SDS-PAGE running buffer
Dissolve 15 g of Tris base (25 mM), 72 g glycine (192 mM) and 5 g SDS (0.1% w/v) in 5 liters of distilled water. Mix until dissolved Store at room temperature.

h) Transfer buffer
Dissolve 15 g of Tris base (25 mM) and 72 g glycine (192 mM) in 4 liters of distilled water. Add 1000 ml methanol (20 % v/v) and mix thoroughly.
Store at 2-8 C.

i) Tris buffered saline (TBS ) pH 7.6
20 ml 1 M Tris HCl pH 7.6 (20 mM)
8 g Sodium chloride (137 mM)
Dilute to 1000 ml with distilled water- check pH. Store at room temperature.

j) TBS-Tween (TBS-T)
Use for wash buffers and diluents.
A 0.1% (v/v) Tween 20 concentration in TBS is suitable for most fluorescent Western blotting work on PVDF membrane but concentration ranging from 0.05% to 1 % may be required to suit your specific needs. Wash buffers should be stored at room temperature.


Flow diagram of procedure

Separate protein sample by electrophoresis
V
Transfer to membrane
V
Block non-specific sites
V
Incubate in primary antibody
V
Incubate in alkaline phosphate linked anti-species antibody
V
Incubate in Vistra ECF substrate
V
Scan

Gel preparation

For 20 ml

Component 8% 10% 15%Do not move the blot during the incubation.Water 9.3 ml 7.9 ml 4.6 ml
30% Acrylamide 5.3 ml 6.7 ml 10 ml
mix (19:1)
1.5 M Tris HCl 5.0 ml 5.0 ml 5.0 ml
pH 8.8
10 % (w/v) SDS 0.2 ml 0.2 ml 0.2 ml
10 % (w/v) AMPS 0.2 ml 0.2 ml 0.2 ml
TEMED 0.012 ml 0.008 ml 0.008 ml

Stacking gel:

Component
Water 5.6 ml
30 % Acrylamide mix (19:1) 1.7 ml
0.5 M Tris HCl pH 6.8 2.5 ml
10 % (w/v) SDS 0.1 ml
10 % (w/v) AMPS 0.1 ml
TEMED 0.01 ml
Mix samples with loading buffer and boil for 5-10 min. After load on the gel and let separate with constant 20 mA. (If two gels 40 mA)

Performing the blotting:

After the electrophoresis build a sandwich out of gel and membrane:
The PVDF membrane should be pre-wetted in methanol for 5 seconds and rinsed in water for 5 min to remove the methanol, then equilibrated in transfer buffer for 10 to 15 min.
Cut out some whatman paper in the same size like gel and membrane.
Sandwich order:
a) two or three whatman paper
b) PVDF membrane
c) Gel
d) Two or three whatman paper
The sandwich should be put in the blotting machine so that the membrane is on the side of the Cathode.

Blocking the membrane

Remove the membrane from the apparatus. Block the nonspecific binding sites by immersing the membrane in 5% blocking reagent in Tris buffered saline Tween 20 (TBS-T) for one hour on an orbital shaker at room temperature
Optimum Tween concentration will vary to suit specific experiments, but a 0.1% Tween 20 concentration is suitable for most fluorescent work on PVDF membrane. Certain experimental situation may require alteration of the time and temperature of the blocking incubation.
Alternatively, membranes may be left in the blocking solution overnight at 2-8 C.

Washing Wash membrane with TBS-T.
Briefly rinse membrane twice with fresh changes of washing buffer. Then wash three times: 20 min, 10 min, 10 min. with fresh changes of washing buffer on an orbital shaker at room temperature
The volume of wash buffer should be as large as possible. 4 ml of buffer per cm2 of membrane is suggested.

Primary antibody During the washing step dilute the primary antibody in TBS-T.
(for 5-alpha reductase: 1:1000 )
Incubate the membrane in the diluted antibody for 1 hour on an orbital shaker at room temperature.
The required dilution of the primary antibody to give the optimum results will vary and should be determined for each antibody used.
When quantifying protein samples linearity can, in some cases, be improved by decreasing the primary antibody concentration. Too high a concentration may lead to saturation of binding sites which will hinder the binding of the secondary antibody and should therefore be avoided. For impure antibodies the addition of blocking reagent to the primary antibody incubation may improve signal to noise ratio.

Washing
s.a.

Secondary antibody
Dilute the anti-rabbit-AP 1:10000 in TBS-T.
Incubate the membrane in diluted antibody for 1 hour on an orbital shaker at room temperature.

Washing
s.a.

Substrate
Use 24 ul substrate per cm2 membrane.
Place a piece of saran wrap on the bench and using tissue to smooth it out ensuring there are no air bubbles or creases in it. Pipette the substrate on the saran wrap and lay the membrane with the protein side on the substrate (ensure that there are no air bubbles)
Leave on the substrate for 5 min at room temperature.

Do not move the blot during the incubation.
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