**5. Appendix A: Microalgae methodology**

*Chlamydomonas reinhardtii* **(Plantae, Chlorophyta)** (Förster et al., 2006). The figure correspond to the species mentioned.

This protocol is an adaption of the one described by Mathesius and co-workers (Grotewold, 2003) that are suited for plant material.


Proteomics in Seaweeds: Ecological Interpretations 39

2. Mix suspension with the same volume of sample buffer containing 0.3% w/v SDS, 1%

3. Denature solution at 100°C for 3 min, cool on ice and treat with DNase/RNase.

4. Wash the pellet with 100% acetone several times and then air-dry it at room

5. Determine protein concentration of the sample (e.g. with a Bradford assay or a BCA

*Gracilaria changii* **(Plantae, Rhodophyta)** (Wong et al., 2006). The figure correspond to the

In this study two protein extraction methods were analysed in two-dimensional gels. The best results were yielded by the phenol/chloroform method, which is detailed below.

1. Grind frozen seaweeds at -70°C into a fine powder with a mortar and pestle in liquid nitrogen. Put approximately 100 mg of the resulting powder into a 1.5 mL tube for a

2. Add 1 mL of TRI reagent (containing phenol and guanidine-isothiocyanate) to 100 mg

3. Store the homogenate for 5 min at room temperature to clarify phases. Reserve the

4. Cover the samples and shake vigorously for 15 seconds. Store the resulting mixture at room temperature for 2-15 min. Centrifuge mixture at 12,000 *g* for 15 min at 4°C. 5. Discard upper aqueous phase containing RNA, and retain interphase and lower red

7. Retain phenol/ethanol supernatant and add 3 volumes of acetone to precipitate

8. Store sample for 10 min at room temperature and sediment the protein precipitate at

9. Discard the phenol/ethanol supernatant and disperse the protein pellet in 0.5 mL of 0.3

10. Add another 0.5 mL aliquot of the guanidine hydrochloride/ethanol/glycerol solution to the sample and store for 10 min at room temperature. Centrifuge the proteins at

phenolic phase and add 200 µL of chloroform per 1 mL of TRI reagent.

proteins, mix by inversion for 10-15 sec to obtain a homogenous solution.

M guanidine hydrochloride in 95% ethanol + 2.5% v/v glycerol.

Precipitate proteins with 10% TCA in 100% Acetone at -70°C for 3 h.

w/v β-mercaptoethanol and 0.05 M Tris-HCl pH 8.0.

assay) and keep at –80°C until used for isoelectric focusing.

**6. Appendix B: Macroalgae methodology** 

of seaweed powder and homogenize the mixture.

phenol-chloroform phase containing DNA and proteins. 6. Add ethanol to the reserved phases in order to precipitate DNA.

temperature for 5 min.

species mentioned.

single extraction.

12,000 *g* for 10 min at 4° C.

8,000 *g* for 5 min.

*Haematococcus pluvialis* **(Plantae, Chlorophyta)** (Wang et al., 2003). The figure correspond to the species mentioned.

In this study a number of key chemical reagents were evaluated, the protocol that yielded the best 2-DE results is detailed below.


*Nannochloropsis oculata* **(Chromista, Ochrophyta)** (Kim et al., 2005). The figure correspond to the species mentioned.

In this study only one method for protein extraction was performed, the details are shown below.

1. Collect *N. oculata* cells by centrifugation at 12,000 x *g* for 10 min. Suspend cell pellet in PBS buffer pH 7.2.

*Haematococcus pluvialis* **(Plantae, Chlorophyta)** (Wang et al., 2003). The figure correspond to

In this study a number of key chemical reagents were evaluated, the protocol that yielded

1. Collect *H. pluvialis* cells by centrifugation at 3,000 x *g* for 5 min and wash the pellet three

2. Resuspend cell pellet in one volume of 50 mM Tris-HCl buffer pH 8.0, 3 mM DTT, 5 mM MgCl2, 10% glycerol, 0.5% PVP, 5 mM Na2-EDTA, 1 mM PMSF, 5 mM benzamidin,

3. Disrupt cells by one passage through a pre-cooled French Press Cell at a pressure of

5. Dialyze the supernatant from the previous centrifugation step against 250 mL of 85% w/v sucrose solution at 4°C for 2 h. Precipitate the dialysate with 9 volumes of icecold 10% w/v TCA in acetone containing 0.07% w/v β-mercaptoethanol at -20°C

6. Centrifuge samples at 15,000 x *g*. Discard supernatant and wash the pellet with acetone

8. Resuspend pellet in solubilization buffer containing 2 M thiourea, 8 M urea, 4% CHAPS, 2 mM TBP, and 0.2% ampholytes (ones suited for the desired 2-DE gel). 9. Determine protein concentration of the sample (e.g. with a Bradford assay or a BCA

*Nannochloropsis oculata* **(Chromista, Ochrophyta)** (Kim et al., 2005). The figure correspond to

In this study only one method for protein extraction was performed, the details are shown

1. Collect *N. oculata* cells by centrifugation at 12,000 x *g* for 10 min. Suspend cell pellet in

5 mM acoproic acid and 1% v/v plant protease inhibitor cocktail.

4. Collect the supernatant and centrifuge at 100,000 *g* for 1 h.

containing 0.07% w/v β-mercaptoethanol to remove TCA.

assay) and keep at –80°C until used for isoelectric focusing.

7. Then, remove residual acetone by air-drying.

20,000 psi. Centrifuge cell lysate at 3,000 *g* for 10 min to pellet cell debris.

the species mentioned.

overnight.

the species mentioned.

PBS buffer pH 7.2.

below.

the best 2-DE results is detailed below.

times with cold deionized water.


#### **6. Appendix B: Macroalgae methodology**

*Gracilaria changii* **(Plantae, Rhodophyta)** (Wong et al., 2006). The figure correspond to the species mentioned.

In this study two protein extraction methods were analysed in two-dimensional gels. The best results were yielded by the phenol/chloroform method, which is detailed below.


Proteomics in Seaweeds: Ecological Interpretations 41

In this study a very simple method for protein extraction was performed, the details are

2. Homogenize seaweed material using a mortar-driven homogenizer in sample lysis solution composed of 7 M urea, 2 M thiourea, containing 4% w/v CHAPS, 1% w/v

7. Determine protein concentration of the sample (e.g., with a Bradford assay or a BCA

*Saccharina japonica* and *Ecklonia cava* **(Chromista, Ochrophyta)** (Yotsukura et al., 2010; 2012).

2. Freeze seaweed material at -80°C before pulverization. Homogenize the tissue in 99.5%

3. The protein pellet is rinsed in 99.5% ethanol and 100% acetone and resuspended in 0.1 M Tris-HCl buffer pH 8.0, 30% sucrose, 2% SDS, 5% β-mercaptoethanol and phenol.

\* If cell lysing is found to be difficult, used a bead beater in order to facilitate the process.

1. Freeze seaweed material at -80°C before pulverization.

DTT, 2% v/v ampholytes and 1 mM benzamidine. 3. Perform freezing and thawing steps for five times for 1 day\*. 4. Extract proteins for 1 h at room temperature with vortexing.

6. Retain soluble fraction and discard insoluble material.

assay) and keep at –80°C until used for isoelectric focusing.

5. Centrifuge mixture at 15,000 *g* for 1 h at 18°C.

The figures correspond to the species mentioned.

1. Remove the salt excess in Milli Q water.

cold ethanol and centrifuged.

shown below.


*Scytosiphon gracilis* and *Ectocarpus siliculosus* **(Chromista, Ochrophyta)** (Contreras et al., 2008). The figures correspond to the species mentioned.

In this method, a major extraction of proteins was obtained in comparison with pervious macroalgae methods described.


*Bostrychia radicans / B. moritziana* **(Plantae, Rhodophyta)** (Kim et al., 2008). The figure correspond to *B. moritziana*.

11. Discard the wash solution and perform two more washes in 1 mL each of the guanidine

12. Perform a final wash in 1 mL of ethanol containing 2.5% glycerol v/v. At the end of the

14. Resolubilize protein pellet in 40 mM Tris buffer pH 8.8 containing 8 M urea, 4% CHAPS

15. Determine protein concentration of the sample (e.g., with a Bradford assay or a BCA

*Scytosiphon gracilis* and *Ectocarpus siliculosus* **(Chromista, Ochrophyta)** (Contreras et al.,

In this method, a major extraction of proteins was obtained in comparison with pervious

3. Homogenize seaweed material using a mortar-driven homogenizer in sample lysis solution composed of 1.5% PVP, 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl pH 7.5, 250 mM EDTA, protease inhibitor cocktail, 2% v/v β-mercaptoethanol and 0.5% w/v CHAPS 4. Equal volume of Tris-HCl pH 7.5-satured phenol is added and the mixture homogenized at 4°C. Then, by centrifugation the upper phase is removed and the lower

5. The proteins in the phenol phase are precipitated by means of ammonium acetate (0.1 M in methanol). The protein pellet obtained by centrifugation is washed in 80% ice-cold

6. Determine protein concentration of the sample and keep at –20°C until used for

*Bostrychia radicans / B. moritziana* **(Plantae, Rhodophyta)** (Kim et al., 2008). The figure

1. Remove the excess salt by rinsing in Milli Q water and 50 mM Tris-HCl pH 8.8.

2. Freeze seaweed material at -80°C before pulverization.

phase is re-extracted using the same volume of phenol.

acetone and cold acetone containing 20 mM DTT.

10 min of ethanol wash, centrifuge the proteins at 8,000 *g* for 5 min. 13. Discard the alcohol and air-dry the pellet for 7-10 min at room temperature.

assay) and keep at –80°C until used for isoelectric focusing.

Hydrochloride/ethanol/glycerol wash solution.

2008). The figures correspond to the species mentioned.

and 2 mM TBP.

macroalgae methods described.

isoelectric focusing.

correspond to *B. moritziana*.

In this study a very simple method for protein extraction was performed, the details are shown below.


\* If cell lysing is found to be difficult, used a bead beater in order to facilitate the process.

*Saccharina japonica* and *Ecklonia cava* **(Chromista, Ochrophyta)** (Yotsukura et al., 2010; 2012). The figures correspond to the species mentioned.


Proteomics in Seaweeds: Ecological Interpretations 43

This work was supported by FONDECYT 11085019 to LC. Additional funding cames from FONDAP 1501-0001 (CONICYT) to the Center for Advanced Studies in Ecology and Biodiversity (CASEB) Program 7. Finally, we are especially grateful to Nicole Ehrenfeld and Javier Tapia for image acquisition and Daniela Thomas, Alejandra Nuñez and Aaron

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**8. References** 


*Porphyra columbina* **(Plantae, Rhodophyta).** The figure correspond to the species mentioned.

This method is an adaptation of the method performed by Contreras et al. 2008, where the details of it are shown below\*.

1. Pulverize 3-5 g of frozen seaweeds at -80°C to a fine powder with a mortar and pestle in liquid nitrogen.


### **7. Acknowledgment**

42 Gel Electrophoresis – Advanced Techniques

4. The solution is vortexed and centrifuged at room temperature and the upper phase

5. The protein pellet obtained by centrifugation is rinsed in 0.1 M ammonium acetate in methanol and 80% acetone, subsequently dried in evaporator and preserved at –80°C

*Porphyra columbina* **(Plantae, Rhodophyta).** The figure correspond to the species mentioned. This method is an adaptation of the method performed by Contreras et al. 2008, where the

1. Pulverize 3-5 g of frozen seaweeds at -80°C to a fine powder with a mortar and pestle in

2. Resuspend pulverized tissue in 5-10 mL of buffer lysis containing 0.5 M Tris-HCl pH 7.5, 0.7 M sucrose, 0.5 M KCl, 250 mM EDTA, 1.5% w/v PVP, 0.5% w/v CHAPS and 2%

3. Add an equal volume of Tris–HCl pH 7.5-saturated phenol and homogenize for 15 min

4. Retain only upper phenol phase containing proteins being careful not to remove the

7. Retain newly only upper phenol phase containing proteins being careful not to remove

10. Centrifuge at 2,000 *g* for 40 min. Discard supernatant and wash pellet with 8 volumes of

13. The proteins pellet is washed in 80% ice-cold acetone and cold acetone containing 20 mM

14. The protein pellet is then washed in ice-cold acetone 80% and ice-acetone 60% in

15. Determine protein concentration of the sample and keep at -20°C until used for isoelectric

5. Add ½ volumes of Tris–HCl pH 7.5-saturated phenol and mix well by inversion.

the interphase and mix with the previously retained upper phase. 8. Add 5 volumes of 0.1 M ammonium acetate on methanol ice-cold.

9. Shake vigorously to mix the solution and leave to precipitate for 4 h at -20°C.

11. Shake vigorously to mix the solution and leave to precipitate for 30 min at -20°C.

v/v β-mercaptoethanol and homogenize for 15 min at 4°C.

at 4° C. Centrifuge the homogenate at 2,000 *g* for 30 min.

6. Centrifuge the homogenate at 2,000 *g* for 20 min.

0.1 M ammonium acetate on methanol ice-cold.

methanol to remove the majority of contaminants.

12. Centrifuge at 2,000 *g* for 30 min.

collected. Agitate solution in 0.1 M ammonium acetate and kept at -20°C.

until protein quantification.

details of it are shown below\*.

liquid nitrogen.

interphase.

DTT.

focusing.

\* Method not yet published.

This work was supported by FONDECYT 11085019 to LC. Additional funding cames from FONDAP 1501-0001 (CONICYT) to the Center for Advanced Studies in Ecology and Biodiversity (CASEB) Program 7. Finally, we are especially grateful to Nicole Ehrenfeld and Javier Tapia for image acquisition and Daniela Thomas, Alejandra Nuñez and Aaron Mann for text editing.

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**3** 

 *Turkey*

**Gel Electrophoresis Based Genetic** 

*1Istanbul Technical University, Environmental Engineering Department, Maslak, Istanbul 2Bogazici University, Institute of Environmental Science, Rumelihisarustu-Bebek, Istanbul* 

Molecular tools in environmental microbiology have been applied extensively in last decades because of the limitations in culture-dependent methods (Amann *et al.*, 1995; Muyzer *et al.*, 1996; Head *et al.*, 1998). Despite isolation techniques are provided detailed knowledge about the single species in terms of morphology, biochemistry, and also genetic (Bitton, 2005), they have important drawbacks. The first one is to find the selective media favoring the desired microbial group. Additionally, isolated species cannot reflect their behaviors in the natural environment. Until today, 19.000 microbial species have been isolated (DSMZ, 2011; http://www.dsmz.de), however it is accepted that this number is only a small portion of real diversity (Amann *et al.*, 1995). Besides, using the molecular tools in natural and engineering systems, we can find the answer to the questions such as 'which species do exist?', 'which species are active?', 'how many microorganisms are there?', which

Microbial ecology studies need identification of species based on a comprehensive classification system that perfectly reflects the evolutionary relations between the microorganisms (Pace, 1996). Zuckerkandl and Pauling (1965) indicated that nucleic acids could document evolutionary history. Due to the pioneering studies, nucleic acids, especially 16S rRNA, are the ultimate biomarkers and hereditary molecules probably because of their essential role in protein synthesis, making them one of the earliest evolutionary functions in all cellular life-forms (Olsen *et al.*, 1986; Pace *et al.*, 1986; Woese, 1987; Stahl *et al.*, 1988). In particular, 16S rRNA and 16S rDNA have been used in

Genetic fingerprinting techniques are one of the most applied molecular tools based on 16S rRNA in microbial ecology studies. These techniques such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), amplified ribosomal DNA restriction (ARDRA) or restriction fragment length polymorphism (RFLP), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP), have been developed for estimation of diversity in ecosystems, screening clone libraries, following the diversity changes with respect to time

phylogenetic analysis and accepted as ideal evolutionary chronometer.

**1. Introduction** 

species do utilize the specific compounds?'.

**Fingerprinting Techniques on** 

Zeynep Cetecioglu1, Orhan Ince1 and Bahar Ince2

**Environmental Ecology** 

