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Molecular Biotechnology
 
October 6 - 17
Paula Veríssimo, Bruno Manadas, Sandra Ribeiro, Rui Cruz

Lab. Biotecnologia Molecular do CNC
Dep. Bioquímica, Universidade de Coimbra

IBMC

Lectures

Lect.1: Cloning strategies

Lect. 2& 3: Expression of cloned genes in prokaryotes

Lect. 4&5: Expression of cloned genes in eukaryotes

Lect. 6: Recombinant protein production

Lect. 7&8: Protein purification

Lect. 9: Protein engineering

Lect. 10: Proteomics

Lect. 11: Protein aggregation and disease

 

Practical course

Protein engineering

Expression, purification and characterisation of recombinant enzymes

 

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

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%
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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