Application of the two-dimensional gel electrophoresis (2DE) protocols which were developed for samples of mammalian origin gives unsatisfactory results when used in samples from marine molluscs. This chapter describes a detailed protocol of 2DE that can be applied to these organisms, especially for Ruditapes decussatus and Bathymodiolus azoricus.info:eu-repo/semantics/publishedVersio

Bebianno, Maria João

Chora, Suze

Roméo, Michèle

English

Sapientia (Univ. do Algarve)

BookID 146080_ChapID 12_Proof# 1 - 15/12/2008 Chapter 12 Analysis of Proteins from Marine Molluscs  Suze  Chora,  Maria  Bebianno , and  Michèle  Roméo  Summary  Application of the two-dimensional gel electrophoresis (2DE) protocols which were developed for sam-ples of mammalian origin gives unsatisfactory results when used in samples from marine molluscs. This chapter describes a detailed protocol of 2DE that can be applied to these organisms, especially for  Rudi-tapes decussatus and  Bathymodiolus azoricus .  Key words: Two-dimensional electrophoresis (2DE)’ ,  Marine bivalves ,  Ruditapes decussatus ,  Bathy-modiolus azoricus .    Marine molluscs, particularly mussels, oysters, and clams, have been used worldwide as bioindicators to assess the impact of pol-lutants in coastal marine ecosystems and their health status. More recently, the discovery in 1997 of large mussel beds in deep sea hydrothermal vents  (1) , particularly in the Mid-Atlantic Ridge, has attracted much scientific attention due to their capacity to live in one of the most extreme environments on earth characterized by high temperature and pressure, low pH, enriched in toxic sul-phide species  (2) , radionuclides, and naturally high bioavailable metal concentrations that would be toxic or even lethal to coastal marine species  (3,  4) . Therefore these species have been consid-ered as models to assess pollution effects in natural contaminated environments  (5) .  The isolation of proteins from marine bivalves from coastal or extreme environments, like deep sea hydrothermal vents, using two-dimensional electrophoresis (2DE) was proved not to be straightforward when using standard protocols developed for 1. Introduction12345678910111213141516171819202122232425262728David Sheehan and Raymond Tyther (eds.), Methods in Molecular Biology, Two Dimensional Electrophoresis Protocols, vol. 519© Humana Press, a part of Springer Science + Business Media, LLC 2009DOI: 10.1007/978-1-59745-281-6_121146080_Sheehan_12.indd   1 12/22/2008   1:06:12 PM2 Chora, Bebianno, and RoméoBookID 146080_ChapID 12_Proof# 1 - 15/12/2008mammalian tissues mainly due to the excess of salt water and the lipid content of some of particular tissues studied. Recently 2DE protocols were developed for bivalve species in particularly for the coastal clam  Ruditapes decussatus and the deep sea hydrother-mal vent mussel  Bathymodiolus azoricus . Two tissues were used: gills and digestive gland. 2DE gels (18 × 18 cm) were able to separate more than 2,000 proteins.  Although the genome of these species is unknown, a number of proteins have already been sequenced. Some relevant proteins were separated from the spots and some of them sequenced. In bivalves, gill and digestive gland tissues usually do not provide good resolution 2DE gels because they may contain exogenous proteins. To overcome this problem in this protocol we describe the best technique for protein separation using 2DE.   IKA, model Ultra-Turrax TD 25, Ettan IPGphor II (GE Health-care), Protean II xi cell (Bio-Rad, Labs). Vertical system.  1.  Homogenization buffer : 10 mM HEPES and 250 mM Sucrose, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM PMSF, 10% protease inhibitors (protease inhibitor tablets, Sigma, Aldrich). DTT, PMSF, and protease inhibitors are added just prior to use. This can be stored at 4°C for 1 week.  2.  Precipitation solution : 10% trichloroacetic acid (TCA) in ace-tone containing 20 mM DTT. DTT is added just prior to use. This can be stored at 4°C for 1 week.  3.  Sample dilution buffer : 7 M urea (GE Healthcare), 2 M thiou-rea (GE Healthcare), amberlite MB-150 1% (Sigma, Aldrich)  (6) . Filter solution through 0.20- m m filter. Add 4% CHAPS (GE Healthcare), 0.8% Pharmalyte (GE Healthcare), 65 mM DTT (GE Healthcare), 10% isopropanol  (6) , and a few grains of bromophenol blue (BPB). Store in 2.5 mL aliquots at −70°C for 2 months.  4.  SDS equilibration buffer : urea 6 M, Tris–HCl, pH 8.8, 75 mM, glycerol 3%, SDS 4%, and a few grains of bromophenol blue (BPB). Store aliquoted. Can be stored at −20°C for 2 months ( see  Note  1 ).  5.  Electrophoresis buffer : SDS: 0.1%, Tris base: 25 mM, Glycine: 192 mM. Store at room temperature.  6.  10% SDS solution : Filter solution through a 0.45- m m filter and store at room temperature. 2. Materials2.1. Equipment2.2. Solutions and Reagents29303132333435363738394041424344454647484950515253545556575859606162636465666768146080_Sheehan_12.indd   2 12/22/2008   1:06:13 PM Analysis of Proteins from Marine Molluscs 3BookID 146080_ChapID 12_Proof# 1 - 15/12/2008 7.  10% ammonium persulphate solution : Prepare just prior to use.  8.  Agarose sealing solution : 0.5% agarose. Add all ingredients into a 500 Erlenmeyer flask. Swirl to disperse. Heat on a heating stirrer until the agarose is completely dissolved ( see  Note  2 ). Dispense 2 mL aliquots and store at room tem-perature.  9.  EDTA 500 mM : store at room temperature.  10.  Immobiline DryStrip, pH 3–10 NL, 18 cm (GE Health-care), immobilized pH-gradient isoelectric focusing gels for the first-dimension separation step.  PdQuest (Bio-Rad, Laboratories, Hercules, CA).   In contamination experiments animals should be acclimated in clean sea water for 7 days prior to the beginning of the experi-ment  (7) ( see  Note  3 ).  1.  Dissect tissues at 4°C, using a scalpel cooled by dipping peri-odically into liquid nitrogen. It is convenient to use a pair of thin cotton gloves during these procedures to avoid transfer-ring heat to the samples.  2.  Collect tissues in 5-mL screw-capped plastic tubes on liquid nitrogen.  3.  When exposed to the cold, the tissues immediately freeze and can subsequently be stored at −70°C or below ( see  Note  4 ).  Having obtained clean samples, it is very important to mince them effectively. Failure to do so will result in selective extraction of protein, which will distort the results of the experiment ( see  Note  5 ). 1.  Suspend each sample in three volumes of homogenization buffer and homogenize in 15-mL tubes, using an Ultra-Turrax IKA-Werke homogenizer at 4°C in a cold room, on ice ( see  Note  6 and Chapter “Difficult Proteins”).  2.  Centrifuge the homogenate at 15,000 ×  g for 2 h at 4°C.  3.  Transfer the supernatant into 2.5-mL Eppendorf tubes for sub-sequent quantification of sample protein ( see Subheading 3.6 ).  4.  Prepare aliquots of sample so that the final quantity of protein is 300  m g. 2.3. Software3. Methods3.1. Preparation of Animals3.2. Protein Sample Preparation3.3. Total Protein Extraction69707172737475767778798081828384858687888990919293949596979899100101102103104105146080_Sheehan_12.indd   3 12/22/2008   1:06:13 PM4 Chora, Bebianno, and RoméoBookID 146080_ChapID 12_Proof# 1 - 15/12/2008 Protein precipitation is an optional step in sample preparation for 2DE. Precipitation is generally employed selectively to separate proteins in the sample from contaminating species such as salts, detergents, nucleic acids, lipids, etc. that might otherwise inter-fere with the 2DE result  (8) . 1.  Suspend each sample in nine volumes of precipitation solution ( see  Note  7 ).  2.  Precipitate proteins for at least 2 h at −20°C.  3.  Pellet proteins by centrifugation at 10,000 ×  g for 30 min.  4.  Wash pellet with cold acetone ( see  Note  8 ).  5.  Remove residual acetone by air drying.  1.  Resuspend the precipitate in the sample dilution buffer. Use 300  m g in 300  m L, vortex, and leave for 30 min.  2.  Centrifuge at 14,000 ×  g for 10 min ( see  Note  9 ).  Quantification of the proteins concentration was carried out using ovalbumin as a protein standard using the Bradford method  (9) .  Isoelectric focusing is an electrophoretic method that separates proteins according to their isoelectric points (pI). For this separa-tion wide-range immobilized pH-gradient (IPG) gels, with pH values ranging from 3 to 10, in 18-cm length strips, were used ( see  Note  10 ).  About 300  m g of total extractable protein can be resolved by IEF on immobiline ® DryStrip, pH 3–10, NL, 18 cm (Pharmacia Biotech): 1.  Pipette the samples (prepared as described in the  Subhead-ing 3.5 ) into each strip holder. Distribute the solution evenly over the channel length and remove any large bubbles.  2.  Carefully remove the cover foil from the Immobiline DryS-trip, starting from the anodic end (+ end).  3.  Carefully place the Immobiline DryStrip into the holder chan-nel, gel-side down ( see  Note  11 ).  4.  Overlay the strip with Immobiline DryStrip Cover Fluid to minimize evaporation and prevent urea crystallization.  5.  Apply the pressure blocks on the underside of the cover to ensure that the Immobiline DryStrip gel maintains good con-tact with the electrodes as the gel swells.  6.  Ensure that the strip holders are properly positioned on the Ettan IPGphor II platform. Use the guide marks along the sides of the platform to position each strip holder and check that the pointed end is over the anode (pointing to the back of the unit) and the blunt end is over the cathode. (Please refer 3.4. Precipitation Procedure3.5. Protein Solubilization3.6. Quantification of Sample Protein3.7. Isoelectric Focusing106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146146080_Sheehan_12.indd   4 12/22/2008   1:06:13 PM Analysis of Proteins from Marine Molluscs 5BookID 146080_ChapID 12_Proof# 1 - 15/12/2008to the Ettan IPGphor II user manual for complete details). Check that both external electrode contacts on the underside of each Strip Holder make metal-to-metal contact with the platform.  7.  Close the Ettan IPGphor II lid. Program the first step of Ettan IPGphor II to make an active rehydration during 12 h under low voltage (20 V) at low current (50  m A/IPG strip), at 20°C ( see  Note  12 ).  8.  Program the other steps of the IEF: 1,000 V, 1 h; 4,000 V 1 h; 8,000 V, 1 h, and 8,000 V, until 50,000 V (~5 h) ( see  Note  13 ).  9.  After IEF is complete, proceed to the second-dimension sepa-ration immediately or store the Immobiline DryStrip gels at −60°C or below in screw-capped tubes.  In the second-dimension separation, where proteins are separated by molecular weight, traditional SDS–PAGE is employed.  1.  Place the IPG strips in individual tubes, with the support film towards the tube wall.  2.  Add 5 mL of SDS equilibration buffer, 100 mg DTT per strip, 0.5  m L EDTA.  3.  Cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process. Equili-brate for 15 min.  4.  Pour off buffer from earlier step and add the appropriate vol-ume of SDS equilibration buffer, 250 mg iodoacetamide, 0.5  m L EDTA to each strip. Again cap or seal the tubes with flex-ible paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for an additional 15 min ( see  Note  14 ).  Cast the 10% polyacrylamide gels using 30% Acrylamide/Bis solution (Bio-Rad Laboratories, Hercules, CA) in 1-mm cassettes ( see  Note  15 ). 1.  Place the strip with the acidic end to the left, gel surface up onto the protruding edge of the longer glass plate.  2.  With a thin plastic ruler, gently push the Immobiline dry strip gel down so that the entire lower edge of the Immobiline DryStrip gel is in contact with the top surface of the gel ( see  Notes  16 and  17 ).  3.  Seal the Immobiline DryStrip gel in place with the agarose sealing solution ( see  Note  18 ).  4.  Insert the cassettes into the tank and pour the electrophoresis buffer to the fill line.  5.  Close the lid and connect the power leads to the power supply. 3.8. Second-Dimen-sion SDS–PAGE3.8.1. Equilibrating Immobiline DryStrip Gels3.8.2. Electrophoresis Using a Vertical Electrophoresis System147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188146080_Sheehan_12.indd   5 12/22/2008   1:06:13 PM6 Chora, Bebianno, and RoméoBookID 146080_ChapID 12_Proof# 1 - 15/12/2008 6.  Electrophoresis is performed at constant current (90 A) in two steps. During the initial migration and stacking period (15 min), the current is 80 V, and during the separation we apply 200 V ( see  Note  19 ).  7.  Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel ( see  Note  20 ).   1.  This is a stock solution. Just prior to use, add DTT or iodoa-cetamide, for first and second equilibration, respectively, and the EDTA 0.1 mM.  2.  Do not allow the solution to boil over.  3.  The animals should have the same size to ensure an equal sample.  4.  It is very important that the tissues never defrost between collection and solubilization in electrophoresis buffer in order to avoid proteolysis.  5.  Direct grinding of frozen tissues in liquid nitrogen does not result in a sufficiently fine mincing for efficient extraction.  6.  Start 30 s with a low speed and two times 15 s in a high speed. In this step the homogenate can be frozen at −70°C.  7.  This approach limits proteolysis and other protein modifica-tions. Protease inhibitors were found to be efficient enough to stop the protease activities in the sample preparation proc-ess. A TCA–acetone treatment was employed in addition to PMSF where acid and organic solvent denature almost all proteins including proteases.  8.  Residual TCA must be removed by extensive washing with acetone because extended exposure to this low pH solution may cause some protein degradation or modification.  9.  This step helps to precipitate remaining salts.  10.  These varied pH intervals allow fine-tuning of each separa-tion strategy to increase first-dimension loading and resolve a greater number of spots from crowded areas, and they are available with strip lengths of 7, 11, 13, 18, and 24 cm. Choose shorter strips, i.e. up to 13 cm, for fast, cost-effective screening, or when the most abundant proteins are of high-est interest. The shortest IPG strips give the fastest results, but their sample load is limited. Use longer strips, i.e. 18- and 24-cm strips, for maximal resolution and loading capacity. Longer strips allow detection of more spots and make it easier 4. Notes189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228146080_Sheehan_12.indd   6 12/22/2008   1:06:13 PM Analysis of Proteins from Marine Molluscs 7BookID 146080_ChapID 12_Proof# 1 - 15/12/2008to select and identify the proteins in the map, but require longer times in both the first- and the second-dimension separations.  11.  To help coat the entire gel, gently lift and lower the strip and slide it back and forth along the surface of the solution. Be care-ful not to trap bubbles under the Immobiline dry strip gel.  12.  The active rehydration facilitates the entry of high-molecular-weight proteins into the strips.  13.  Many factors affect the amount of time required for complete focusing, and each specific set of conditions, e.g. sample and rehydration solution composition, Immobiline dry strip gel length, and pH gradient. Ramping the voltage slowly while the sample is entering the IPG strip improve results.  14.  Be consistent with the timing of the equilibration steps.  15.  The composition of this gel should be selected to resolve proteins in the MW range of interest. Thinner gels stain and destain more quickly and generally give less background staining. Thicker gels have a higher protein capacity. Thicker gels are also less fragile and easier to handle.  16.  Ensure that no air bubbles are trapped between the Immo-biline drystrip gel and the slab gel surface.  17.  The MW marker proteins can be applied to a paper piece; then pick up the application piece with forceps and apply to the top surface of the gel next to one end of the Immobiline DryStrip gel.  18.  The agarose sealing solution prevents the Immobiline dry strip gel from moving or floating in the electrophoresis buffer.  19.  For these vertical systems, cooling is optional. However, tem-perature control improves gel-to-gel reproducibility, espe-cially if the ambient temperature of the laboratory fluctuates significantly. For best results, gels should be run at 25°C.  20.  After this step silver staining is the most sensitive staining technique ( see Chapter “Silver Staining of Proteins in 2DE Gels”). For protein identification we use PDQuest, Bio-Rad, Laboratories, Hercules, CA.  References  1 .   Edmond ,  J.M. ,  Measures ,  C. ,  Magum ,  B.M. ,  Grant ,  B. ,  Sclater ,  F.R. ,  et al .  (1979)  On the formation of metal-rich deposits at ridge crests .  Earth and Planetary Science Letter  46 ,  19 – 30  2 .   Blum ,  J. , and  Fridovich ,  I.  (1984)  Enzymatic defenses against oxygen toxicity in the hydro-thermal vent animals  Riftia pachyptila and  Calyptogena magnifica .  Archives of Biochemis-try and Biophysics  228 ,  617 – 620  3 .   Desbruyères ,  D. ,  Biscoito ,  M. ,  Caprais ,  J.C. ,  Colaço ,  A. ,  Comtet ,  T. ,  Crassous ,  P. ,  et al .  (2001)  Variations in deep-sea hydrothermal vent communities on the mid-atlantic ridge near the Azores plateau .  Deep Sea Research Part I: Oceanographic Research Paper  48 ,  1325 – 1346 229230231232233234235236237238239240241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278146080_Sheehan_12.indd   7 12/22/2008   1:06:13 PM8 Chora, Bebianno, and RoméoBookID 146080_ChapID 12_Proof# 1 - 15/12/2008 4 .   Géret ,  F. ,  Rainglet ,  F. , and  Cosson ,  R.P.  (1998)  Comparison between isolation protocols com-monly used for the purification of mollusc met-allothioneins .  Marine Environmental Research  46 ,  545 – 550  5 .   Bebianno ,  M.J. ,  Company ,  R. ,  Serafim ,  A. ,  Camus ,  L. ,  Cosson ,  R. , and  Fiala-Médoni ,  A.  (2005)  Antioxidant systems and lipid peroxida-tion in  Bathymodiolus Azoricus from mid Atlan-tic ridge hydrothermal vent fields .  Aquatic Toxicology  75 ,  354 – 373  6 .   Starita-Geribaldi ,  M. ,  Roux ,  F. ,  Garin ,  J. ,  Chevallier ,  D. ,  Fénichel ,  P. , and  Pointis ,  G. ,  (2003)  Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis .  Proteomics  3 ,  1611 – 1619  7 .   Coelho ,  M. R. ,  Bebianno ,  M.J. , and  Langston ,  W.J.  (2002) .  Routes of TBT uptake in the clam  Rudi-tapes decussates – Food as vector of TBT uptake .  Marine Environmental Research  54 ,  193 – 207 8.  Bjellqvist, B., Laird, N., McDowell, M., Olsson, I., and Westermeier, R. (1998). 2–D Electrophoresis, preinciples and Methods. Amersham-Biosciences 100 pp 9 .   Bradford ,  M.  (1976)  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding .  Analytical Biochemistry  72 ,  248 – 254 279280281282283284285286287288289290291292293294146080_Sheehan_12.indd   8 12/22/2008   1:06:13 PM

Analysis of proteins from marine molluscs

Suze Chora

Maria Bebianno

Michèle Roméo

Crossref

Analysis of Proteins from Marine Molluscs

Bebianno, Maria

Universidade do Algarve

BookID 146080_ChapID 12_Proof# 1 - 15/12/2008
 Chapter 12
 Analysis of Proteins from Marine Molluscs 
 Suze  Chora,  Maria  Bebianno , and  Michèle  Roméo 
 Summary 
 Application of the two-dimensional gel electrophoresis (2DE) protocols which were developed for sam-
ples of mammalian origin gives unsatisfactory results when used in samples from marine molluscs. This 
chapter describes a detailed protocol of 2DE that can be applied to these organisms, especially for  Rudi-
tapes decussatus and  Bathymodiolus azoricus . 
 Key words: Two-dimensional electrophoresis (2DE)’ ,  Marine bivalves ,  Ruditapes decussatus ,  Bathy-
modiolus azoricus .  
 
 Marine molluscs, particularly mussels, oysters, and clams, have 
been used worldwide as bioindicators to assess the impact of pol-
lutants in coastal marine ecosystems and their health status. More 
recently, the discovery in 1997 of large mussel beds in deep sea 
hydrothermal vents  (1) , particularly in the Mid-Atlantic Ridge, 
has attracted much scientific attention due to their capacity to live 
in one of the most extreme environments on earth characterized 
by high temperature and pressure, low pH, enriched in toxic sul-
phide species  (2) , radionuclides, and naturally high bioavailable 
metal concentrations that would be toxic or even lethal to coastal 
marine species  (3,  4) . Therefore these species have been consid-
ered as models to assess pollution effects in natural contaminated 
environments  (5) . 
 The isolation of proteins from marine bivalves from coastal or 
extreme environments, like deep sea hydrothermal vents, using 
two-dimensional electrophoresis (2DE) was proved not to be 
straightforward when using standard protocols developed for 
1. Introduction
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David Sheehan and Raymond Tyther (eds.), Methods in Molecular Biology, Two Dimensional Electrophoresis Protocols, vol. 519
© Humana Press, a part of Springer Science + Business Media, LLC 2009
DOI: 10.1007/978-1-59745-281-6_12
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2 Chora, Bebianno, and Roméo
BookID 146080_ChapID 12_Proof# 1 - 15/12/2008
mammalian tissues mainly due to the excess of salt water and the 
lipid content of some of particular tissues studied. Recently 2DE 
protocols were developed for bivalve species in particularly for 
the coastal clam  Ruditapes decussatus and the deep sea hydrother-
mal vent mussel  Bathymodiolus azoricus . Two tissues were used: 
gills and digestive gland. 2DE gels (18 × 18 cm) were able to 
separate more than 2,000 proteins. 
 Although the genome of these species is unknown, a number 
of proteins have already been sequenced. Some relevant proteins 
were separated from the spots and some of them sequenced. In 
bivalves, gill and digestive gland tissues usually do not provide 
good resolution 2DE gels because they may contain exogenous 
proteins. To overcome this problem in this protocol we describe 
the best technique for protein separation using 2DE. 
 
 IKA, model Ultra-Turrax TD 25, Ettan IPGphor II (GE Health-
care), Protean II xi cell (Bio-Rad, Labs). Vertical system. 
 1.  Homogenization buffer : 10 mM HEPES and 250 mM Sucrose, 
1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM PMSF, 10% 
protease inhibitors (protease inhibitor tablets, Sigma, Aldrich). 
DTT, PMSF, and protease inhibitors are added just prior to 
use. This can be stored at 4°C for 1 week. 
 2.  Precipitation solution : 10% trichloroacetic acid (TCA) in ace-
tone containing 20 mM DTT. DTT is added just prior to use. 
This can be stored at 4°C for 1 week. 
 3.  Sample dilution buffer : 7 M urea (GE Healthcare), 2 M thiou-
rea (GE Healthcare), amberlite MB-150 1% (Sigma, Aldrich) 
 (6) . Filter solution through 0.20- m m filter. Add 4% CHAPS 
(GE Healthcare), 0.8% Pharmalyte (GE Healthcare), 65 mM 
DTT (GE Healthcare), 10% isopropanol  (6) , and a few grains 
of bromophenol blue (BPB). Store in 2.5 mL aliquots at 
−70°C for 2 months. 
 4.  SDS equilibration buffer : urea 6 M, Tris–HCl, pH 8.8, 75 
mM, glycerol 3%, SDS 4%, and a few grains of bromophenol 
blue (BPB). Store aliquoted. Can be stored at −20°C for 2 
months ( see  Note  1 ). 
 5.  Electrophoresis buffer : SDS: 0.1%, Tris base: 25 mM, Glycine: 
192 mM. Store at room temperature. 
 6.  10% SDS solution : Filter solution through a 0.45- m m filter and 
store at room temperature. 
2. Materials
2.1. Equipment
2.2. Solutions 
and Reagents
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 Analysis of Proteins from Marine Molluscs 3
BookID 146080_ChapID 12_Proof# 1 - 15/12/2008
 7.  10% ammonium persulphate solution : Prepare just prior to 
use. 
 8.  Agarose sealing solution : 0.5% agarose. Add all ingredients 
into a 500 Erlenmeyer flask. Swirl to disperse. Heat on a 
heating stirrer until the agarose is completely dissolved ( see 
 Note  2 ). Dispense 2 mL aliquots and store at room tem-
perature. 
 9.  EDTA 500 mM : store at room temperature. 
 10.  Immobiline DryStrip, pH 3–10 NL, 18 cm (GE Health-
care), immobilized pH-gradient isoelectric focusing gels for 
the first-dimension separation step. 
 PdQuest (Bio-Rad, Laboratories, Hercules, CA). 
 
 In contamination experiments animals should be acclimated in 
clean sea water for 7 days prior to the beginning of the experi-
ment  (7) ( see  Note  3 ). 
 1.  Dissect tissues at 4°C, using a scalpel cooled by dipping peri-
odically into liquid nitrogen. It is convenient to use a pair of 
thin cotton gloves during these procedures to avoid transfer-
ring heat to the samples. 
 2.  Collect tissues in 5-mL screw-capped plastic tubes on liquid 
nitrogen. 
 3.  When exposed to the cold, the tissues immediately freeze and 
can subsequently be stored at −70°C or below ( see  Note  4 ). 
 Having obtained clean samples, it is very important to mince 
them effectively. Failure to do so will result in selective extraction 
of protein, which will distort the results of the experiment ( see 
 Note  5 ).
 1.  Suspend each sample in three volumes of homogenization 
buffer and homogenize in 15-mL tubes, using an Ultra-Turrax 
IKA-Werke homogenizer at 4°C in a cold room, on ice ( see 
 Note  6 and Chapter “Difficult Proteins”). 
 2.  Centrifuge the homogenate at 15,000 ×  g for 2 h at 4°C. 
 3.  Transfer the supernatant into 2.5-mL Eppendorf tubes for sub-
sequent quantification of sample protein ( see Subheading 3.6 ). 
 4.  Prepare aliquots of sample so that the final quantity of protein 
is 300  m g. 
2.3. Software
3. Methods
3.1. Preparation 
of Animals
3.2. Protein Sample 
Preparation
3.3. Total Protein 
Extraction
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4 Chora, Bebianno, and Roméo
BookID 146080_ChapID 12_Proof# 1 - 15/12/2008
 Protein precipitation is an optional step in sample preparation for 
2DE. Precipitation is generally employed selectively to separate 
proteins in the sample from contaminating species such as salts, 
detergents, nucleic acids, lipids, etc. that might otherwise inter-
fere with the 2DE result  (8) .
 1.  Suspend each sample in nine volumes of precipitation solution 
( see  Note  7 ). 
 2.  Precipitate proteins for at least 2 h at −20°C. 
 3.  Pellet proteins by centrifugation at 10,000 ×  g for 30 min. 
 4.  Wash pellet with cold acetone ( see  Note  8 ). 
 5.  Remove residual acetone by air drying. 
 1.  Resuspend the precipitate in the sample dilution buffer. Use 
300  m g in 300  m L, vortex, and leave for 30 min. 
 2.  Centrifuge at 14,000 ×  g for 10 min ( see  Note  9 ). 
 Quantification of the proteins concentration was carried out using 
ovalbumin as a protein standard using the Bradford method  (9) . 
 Isoelectric focusing is an electrophoretic method that separates 
proteins according to their isoelectric points (pI). For this separa-
tion wide-range immobilized pH-gradient (IPG) gels, with pH 
values ranging from 3 to 10, in 18-cm length strips, were used 
( see  Note  10 ). 
 About 300  m g of total extractable protein can be resolved by 
IEF on immobiline ® DryStrip, pH 3–10, NL, 18 cm (Pharmacia 
Biotech):
 1.  Pipette the samples (prepared as described in the  Subhead-
ing 3.5 ) into each strip holder. Distribute the solution evenly 
over the channel length and remove any large bubbles. 
 2.  Carefully remove the cover foil from the Immobiline DryS-
trip, starting from the anodic end (+ end). 
 3.  Carefully place the Immobiline DryStrip into the holder chan-
nel, gel-side down ( see  Note  11 ). 
 4.  Overlay the strip with Immobiline DryStrip Cover Fluid to 
minimize evaporation and prevent urea crystallization. 
 5.  Apply the pressure blocks on the underside of the cover to 
ensure that the Immobiline DryStrip gel maintains good con-
tact with the electrodes as the gel swells. 
 6.  Ensure that the strip holders are properly positioned on the 
Ettan IPGphor II platform. Use the guide marks along the 
sides of the platform to position each strip holder and check 
that the pointed end is over the anode (pointing to the back of 
the unit) and the blunt end is over the cathode. (Please refer 
3.4. Precipitation 
Procedure
3.5. Protein 
Solubilization
3.6. Quantification 
of Sample Protein
3.7. Isoelectric 
Focusing
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to the Ettan IPGphor II user manual for complete details). 
Check that both external electrode contacts on the underside 
of each Strip Holder make metal-to-metal contact with the 
platform. 
 7.  Close the Ettan IPGphor II lid. Program the first step of Ettan 
IPGphor II to make an active rehydration during 12 h under 
low voltage (20 V) at low current (50  m A/IPG strip), at 20°C 
( see  Note  12 ). 
 8.  Program the other steps of the IEF: 1,000 V, 1 h; 4,000 V 1 h; 
8,000 V, 1 h, and 8,000 V, until 50,000 V (~5 h) ( see  Note  13 ). 
 9.  After IEF is complete, proceed to the second-dimension sepa-
ration immediately or store the Immobiline DryStrip gels at 
−60°C or below in screw-capped tubes. 
 In the second-dimension separation, where proteins are separated 
by molecular weight, traditional SDS–PAGE is employed. 
 1.  Place the IPG strips in individual tubes, with the support film 
towards the tube wall. 
 2.  Add 5 mL of SDS equilibration buffer, 100 mg DTT per strip, 
0.5  m L EDTA. 
 3.  Cap or seal the tubes with flexible paraffin film and place them 
on their sides on a rocker for the equilibration process. Equili-
brate for 15 min. 
 4.  Pour off buffer from earlier step and add the appropriate vol-
ume of SDS equilibration buffer, 250 mg iodoacetamide, 0.5 
 m L EDTA to each strip. Again cap or seal the tubes with flex-
ible paraffin film and place them on their sides on a rocker for 
the equilibration process. Equilibrate for an additional 15 min 
( see  Note  14 ). 
 Cast the 10% polyacrylamide gels using 30% Acrylamide/Bis 
solution (Bio-Rad Laboratories, Hercules, CA) in 1-mm cassettes 
( see  Note  15 ).
 1.  Place the strip with the acidic end to the left, gel surface up 
onto the protruding edge of the longer glass plate. 
 2.  With a thin plastic ruler, gently push the Immobiline dry strip 
gel down so that the entire lower edge of the Immobiline 
DryStrip gel is in contact with the top surface of the gel ( see 
 Notes  16 and  17 ). 
 3.  Seal the Immobiline DryStrip gel in place with the agarose 
sealing solution ( see  Note  18 ). 
 4.  Insert the cassettes into the tank and pour the electrophoresis 
buffer to the fill line. 
 5.  Close the lid and connect the power leads to the power supply. 
3.8. Second-Dimen-
sion SDS–PAGE
3.8.1. Equilibrating 
Immobiline DryStrip Gels
3.8.2. Electrophoresis 
Using a Vertical 
Electrophoresis System
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 6.  Electrophoresis is performed at constant current (90 A) in 
two steps. During the initial migration and stacking period 
(15 min), the current is 80 V, and during the separation we 
apply 200 V ( see  Note  19 ). 
 7.  Stop electrophoresis when the dye front is approximately 1 
mm from the bottom of the gel ( see  Note  20 ). 
 
 1.  This is a stock solution. Just prior to use, add DTT or iodoa-
cetamide, for first and second equilibration, respectively, and 
the EDTA 0.1 mM. 
 2.  Do not allow the solution to boil over. 
 3.  The animals should have the same size to ensure an equal 
sample. 
 4.  It is very important that the tissues never defrost between 
collection and solubilization in electrophoresis buffer in 
order to avoid proteolysis. 
 5.  Direct grinding of frozen tissues in liquid nitrogen does not 
result in a sufficiently fine mincing for efficient extraction. 
 6.  Start 30 s with a low speed and two times 15 s in a high 
speed. In this step the homogenate can be frozen at −70°C. 
 7.  This approach limits proteolysis and other protein modifica-
tions. Protease inhibitors were found to be efficient enough 
to stop the protease activities in the sample preparation proc-
ess. A TCA–acetone treatment was employed in addition to 
PMSF where acid and organic solvent denature almost all 
proteins including proteases. 
 8.  Residual TCA must be removed by extensive washing with 
acetone because extended exposure to this low pH solution 
may cause some protein degradation or modification. 
 9.  This step helps to precipitate remaining salts. 
 10.  These varied pH intervals allow fine-tuning of each separa-
tion strategy to increase first-dimension loading and resolve 
a greater number of spots from crowded areas, and they 
are available with strip lengths of 7, 11, 13, 18, and 24 cm. 
Choose shorter strips, i.e. up to 13 cm, for fast, cost-effective 
screening, or when the most abundant proteins are of high-
est interest. The shortest IPG strips give the fastest results, 
but their sample load is limited. Use longer strips, i.e. 18- and 
24-cm strips, for maximal resolution and loading capacity. 
Longer strips allow detection of more spots and make it easier 
4. Notes
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 Analysis of Proteins from Marine Molluscs 7
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to select and identify the proteins in the map, but require 
longer times in both the first- and the second-dimension 
separations. 
 11.  To help coat the entire gel, gently lift and lower the strip and 
slide it back and forth along the surface of the solution. Be care-
ful not to trap bubbles under the Immobiline dry strip gel. 
 12.  The active rehydration facilitates the entry of high-molecular-
weight proteins into the strips. 
 13.  Many factors affect the amount of time required for complete 
focusing, and each specific set of conditions, e.g. sample and 
rehydration solution composition, Immobiline dry strip gel 
length, and pH gradient. Ramping the voltage slowly while 
the sample is entering the IPG strip improve results. 
 14.  Be consistent with the timing of the equilibration steps. 
 15.  The composition of this gel should be selected to resolve 
proteins in the MW range of interest. Thinner gels stain and 
destain more quickly and generally give less background 
staining. Thicker gels have a higher protein capacity. Thicker 
gels are also less fragile and easier to handle. 
 16.  Ensure that no air bubbles are trapped between the Immo-
biline drystrip gel and the slab gel surface. 
 17.  The MW marker proteins can be applied to a paper piece; 
then pick up the application piece with forceps and apply to 
the top surface of the gel next to one end of the Immobiline 
DryStrip gel. 
 18.  The agarose sealing solution prevents the Immobiline dry strip 
gel from moving or floating in the electrophoresis buffer. 
 19.  For these vertical systems, cooling is optional. However, tem-
perature control improves gel-to-gel reproducibility, espe-
cially if the ambient temperature of the laboratory fluctuates 
significantly. For best results, gels should be run at 25°C. 
 20.  After this step silver staining is the most sensitive staining 
technique ( see Chapter “Silver Staining of Proteins in 2DE 
Gels”). For protein identification we use PDQuest, Bio-Rad, 
Laboratories, Hercules, CA. 
 References 
 1 .   Edmond ,  J.M. ,  Measures ,  C. ,  Magum ,  B.M. , 
 Grant ,  B. ,  Sclater ,  F.R. ,  et al .  (1979)  On 
the formation of metal-rich deposits at ridge 
crests .  Earth and Planetary Science Letter  46 , 
 19 – 30 
 2 .   Blum ,  J. , and  Fridovich ,  I.  (1984)  Enzymatic 
defenses against oxygen toxicity in the hydro-
thermal vent animals  Riftia pachyptila and 
 Calyptogena magnifica .  Archives of Biochemis-
try and Biophysics  228 ,  617 – 620 
 3 .   Desbruyères ,  D. ,  Biscoito ,  M. ,  Caprais ,  J.C. , 
 Colaço ,  A. ,  Comtet ,  T. ,  Crassous ,  P. ,  et al . 
 (2001)  Variations in deep-sea hydrothermal 
vent communities on the mid-atlantic ridge near 
the Azores plateau .  Deep Sea Research Part I: 
Oceanographic Research Paper  48 ,  1325 – 1346 
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 4 .   Géret ,  F. ,  Rainglet ,  F. , and  Cosson ,  R.P.  (1998) 
 Comparison between isolation protocols com-
monly used for the purification of mollusc met-
allothioneins .  Marine Environmental Research 
 46 ,  545 – 550 
 5 .   Bebianno ,  M.J. ,  Company ,  R. ,  Serafim ,  A. , 
 Camus ,  L. ,  Cosson ,  R. , and  Fiala-Médoni ,  A. 
 (2005)  Antioxidant systems and lipid peroxida-
tion in  Bathymodiolus Azoricus from mid Atlan-
tic ridge hydrothermal vent fields .  Aquatic 
Toxicology  75 ,  354 – 373 
 6 .   Starita-Geribaldi ,  M. ,  Roux ,  F. ,  Garin ,  J. , 
 Chevallier ,  D. ,  Fénichel ,  P. , and  Pointis ,  G. , 
 (2003)  Development of narrow immobilized 
pH gradients covering one pH unit for human 
seminal plasma proteomic analysis .  Proteomics 
 3 ,  1611 – 1619 
 7 .   Coelho ,  M. R. ,  Bebianno ,  M.J. , and  Langston ,  W.J. 
 (2002) .  Routes of TBT uptake in the clam  Rudi-
tapes decussates – Food as vector of TBT uptake . 
 Marine Environmental Research  54 ,  193 – 207 
8.  Bjellqvist, B., Laird, N., McDowell, M., Olsson, I., 
and Westermeier, R. (1998). 2–D Electrophoresis, 
preinciples and Methods. Amersham-Biosciences 
100 pp
 9 .   Bradford ,  M.  (1976)  A rapid and sensitive 
method for the quantitation of microgram 
quantities of protein utilizing the principle of 
protein-dye binding .  Analytical Biochemistry 
 72 ,  248 – 254 
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Analysis of proteins from marine molluscs

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