Name: spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis
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The protocol was adopted from a previous publication11. T. Z., D. Ch., and J. &#268;. were supported by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (NPU I), grant number LO1415. J. &#268;. was also supported by GA CR, Grant number 18-24397S. Access to instruments and other facilities was supported by the Czech research infrastructure for systems biology C4SYS (project no LM2015055). M. A. S. was supported by a grant from the Russian Science Foundation [no. 14-14-00904].

1. Cyanobacteria Cultivation

<ol>
	<li>Cultivate Synechocystis cells in Erlenmeyer flasks or in photobioreactors10,19 in buffered BG11 medium20 to maintain a pH of &#60; 10 (e.g., using 17 mM HEPES10). 
	NOTE: Standard cultivation conditions require a controlled temperature (typically, 30 &#176;C, the optimal temperature is 35 &#176;C)21, illumination (typical...

<ol>
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Sequence determination of the entire genome and assignment of potential protein-coding regions.</a> DNA Research. 3, 109-136 (1996).</li><li>Grigorieva, G., Shestakov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+in+the+cyanobacterium+Synechocystis+sp+6803.">Transformation in the cyanobacterium Synechocystis sp 6803.</a> FEMS Microbiology Letters. 13 (4), 367-370 (1982).</li><li>Zav&#345;el, T., Sinetova, M. A., B&#250;zov&#225;, D., Liter&#225;kov&#225;, P., &#268;erven&#253;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+model+cyanobacterium+Synechocystis+sp:+PCC+6803+autotrophic+growth+in+a+flat-panel+photobioreactor.">Characterization of a model cyanobacterium Synechocystis sp: PCC 6803 autotrophic growth in a flat-panel photobioreactor.</a> Engineering in Life Sciences. 15 (1), (2015).</li><li>Zav&#345;el, T., O&#269;en&#225;&#353;ov&#225;, P., &#268;erven&#253;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+Synechocystis+sp.+PCC+6803+substrains+reveals+differences+in+sensitivity+to+abiotic+stress.">Phenotypic characterization of Synechocystis sp. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisomes+from+Cyanobacteria.">Phycobilisomes from Cyanobacteria.</a> Handbook on Cyanobacteria: Biochemistry, Biotechnology and Applications. , 105-160 (2009).</li><li>Six, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+evolution+of+phycobilisomes+in+marine+Synechococcus+spp.:+A+comparative+genomics+study.">Diversity and evolution of phycobilisomes in marine Synechococcus spp.: A comparative genomics study.</a> Genome Biology. 8 (12), (2007).</li><li>Sinetova, M. A., &#268;erven&#253;, J., Zav&#345;el, T., Nedbal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+dynamics+and+constraints+of+batch+culture+growth+of+the+cyanobacterium+Cyanothece+sp.+ATCC+51142.">On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142.</a> Journal of Biotechnology. 162 (1), (2012).</li><li>Stanier, R. Y., Kunisawa, R., Mandel, M., Cohen-Bazire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+unicellular+blue-green+algae+(order+Chroococcales).">Purification and properties of unicellular blue-green algae (order Chroococcales).</a> Bacteriological Reviews. 35 (2), 171-205 (1971).</li><li>Zav&#345;el, T., Sinetova, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photobioreactor+system+for+precision+cultivation+of+photoautotrophic+microorganisms+and+for+high-content+analysis+of+suspension+dynamics.">A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics.</a> Biotechnology and Bioengineering. 100 (5), 902-910 (2008).</li><li>Zav&#345;el, T., Knoop, H., Steuer, R., Jones, P. R., &#268;erven&#253;, J., Trt&#237;lek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+evaluation+of+ethylene+production+in+the+recombinant+cyanobacterium+Synechocystis+sp.+PCC+6803+harboring+the+ethylene-forming+enzyme+by+membrane+inlet+mass+spectrometry.">A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp. PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry.</a> Bioresource Technology. 202, 142-151 (2016).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Lawrenz, E., Fedewa, E. J., Richardson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+protocols+for+the+quantification+of+phycobilins+in+aqueous+phytoplankton+extracts.">Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts.</a> Journal of Applied Phycology. 23 (5), 865-871 (2011).</li><li>Lea-Smith, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisome-Deficient+Strains+of+Synechocystis+sp.+PCC+6803+Have+Reduced+Size+and+Require+Carbon-Limiting+Conditions+to+Exhibit+Enhanced+Productivity.">Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.</a> Plant Physiology. 165 (2), 705-714 (2014).</li><li>Seo, Y. 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This protocol describes a simple, fast, and reproducible method for the quantification of phycobiliprotein content in the model cyanobacterium Synechocystis. Several methods of cell homogenization, protein extraction, and phycocyanin and allophycocyanin quantification are compared, and the final protocol represents a combination of the optimal steps of every single procedure. As representative data, the content of phycobiliproteins was quantified in Synechocystis cells under increasing light intensity. ...

fPhycobiliproteins are water-soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria (Cyanophyta) and several eukaryotic taxa (Glaucophyta, Rhodophyta, and Cryptophyta)1. They occur mainly as supramolecular complexes called phycobilisomes and they are typically attached to the surface of the photosynthetic membranes on the stromal side, with the exception of Cryptophyta, where the phycobiliproteins are localized in the thylakoid lumen2. Four types of phycobiliproteins have been identified up to date: the core allophycocyanin, and the peripheral phycocyanin, phycoerythrin, and phycoerythrocyanin1. As the main light-harvesting complexes, phycobilisomes represent one of the crucial factors of algae and cyanobacteria mass cultures productivity. It has been demonstrated that phycobilisomes truncation can enhance biomass accumulation under strong light3. On the other hand, under modest or low irradiance, the antenna truncation resulted in growth rates and biomass accumulation reduction3,4. Phycobiliproteins are commercially used as food colorants, pharmaceuticals, and food additives, in the cosmetic industry, and as fluorescence probes with applications in flow cytometry, fluorescent immunoassays, and fluorescence microscopy5.
This protocol focuses on the quantitative determination of phycobiliproteins in the model cyanobacterium Synechocystis. Cyanobacteria are the earliest oxygenic photosynthetic autotrophs; they have been forming the Earth&#39;s biosphere for more than 2.4 billion years6. They play a crucial role in global biogeochemical cycles of nitrogen, carbon, oxygen, and other elements. Among cyanobacteria, a unicellular strain Synechocystis gained a unique position since it was the first cyanobacterium with the entire genome sequenced7,8, it is naturally transformable by exogenous DNA9, and it performs stable and relatively fast growth10,11. In Synechocystis, the core antenna component, allophycocyanin, is associated with the integral membrane proteins, and the attached phycocyanin is located on the thylakoid membrane periphery.
Several methods for phycobiliprotein extraction and quantification are compared within this protocol. The final extraction procedure was successfully applied to Synechocystis, as well as to other cyanobacteria strains, including Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., and it was also successfully applied to red algae Porphyridium cruentum. Therefore, the method developed in this protocol can be considered as a universal method for phycobiliprotein extraction. Even though some of the tested extraction methods resulted in higher total protein yields, the here described extraction procedure provided the highest phycobiliprotein yields together with the lowest content of chlorophyll a residue in the phycobiliprotein extract. Reducing the content of chlorophyll a was essential for the correct phycocyanin and allophycocyanin spectrophotometric quantification.
The phycobiliprotein absorption spectra can vary significantly among various algae and cyanobacteria species12,13,14,15,16,17 and even among several strains of a single cyanobacteria genus18. Therefore, the specific wavelengths and absorption coefficients as used for the determination of phycocyanin and allophycocyanin in Synechocystis are not generally applicable to other strains. Additionally, Synechocystis does not contain phycoerythrin and phycoerythrocyanin that can be found in some other algae and cyanobacteria. For the purpose of the determination of phycobiliproteins in strains other than Synechocystis, it is recommended to evaluate the spectrophotometric equations for each strain individually.
Although the protocol contains two longer steps (overnight freeze-drying of the cellular pellets and 1-hour protein extraction), the total labor time for the phycobiliproteins quantification is no longer than 2 hours.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Synechocystis sp. PCC 6803</td>
			<td style="width:417px;">Institut Pasteur, Paris, France</td>
			<td style="width:137px;">6803</td>
			<td style="width:627px;">Cyanobacterium strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roti-CELL PBS</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td>9143.1</td>
			<td>Phosphate-Buffered Saline (PBS) solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf safe-lock tubes&#160;</td>
			<td style="width:417px;">Eppendorf, Hamburk, Germany</td>
			<td>30120086</td>
			<td>Safe-lock tubes 1.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR 80-Place Storage System</td>
			<td style="width:417px;">VWR International, Radnor, Pennsylvania, USA</td>
			<td>30128-282</td>
			<td>Holder for safe-lock tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAININ 100 &#181;l -1000 &#181;l&#160;</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>17014382</td>
			<td>Pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GP-LTS-A-1000&#181;L-/F-768/8</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>30389272</td>
			<td>Pipette tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotina 420R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td>4701</td>
			<td>Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LCexv 4010</td>
			<td>Liebherr, Bulle, Switzerland</td>
			<td>9005382197172</td>
			<td>Refrigerator and freezer -20&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Revco ExF -86&#176;C Upright Ultra-Low Temperature Freezer</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">EXF24086V&#160;</td>
			<td>Freezer -80&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolSafe</td>
			<td>LaboGene, Liller&#248;d, Denmark</td>
			<td>7.001.000.615</td>
			<td>Freeze dryer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-2600</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>UV-2600</td>
			<td>Spectrophotometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma absorption cuvettes, semi Micro</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z600288&#160;</td>
			<td>VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silamat S6</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td>602286WU</td>
			<td>Homogenizer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solid-glass beads</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z273627</td>
			<td>Glass bead of the diameter 2&#160;mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPA225D-0CE</td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>SECURA225D-1OBR</td>
			<td>Analytical balances</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-Phycocyanin from Spirulina sp.&#160;</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P2172</td>
			<td>Phycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allophycocyanin</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>A7472</td>
			<td>Allophycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bicinchoninic Acid Kit&#160;</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">BCA1, B9643</td>
			<td>Complete kit for total proteins determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">AlgaeTron&#160;</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">AG 130-ECO&#160;</td>
			<td>Cultivation chamber for E. flasks, with controllable light and atmosphere</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Photobioreactor</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">FMT-150</td>
			<td>Cultivation equipment for cyanobacteria and algae with completely controllable environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer&#160;</td>
			<td style="width:417px;">Nexcelom Bioscience, Lawrence, Massachusetts, USA</td>
			<td style="width:137px;">Auto M10</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Corning 15 mL centrifuge tubes</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">CLS430791&#160;</td>
			<td>15 ml Centrifuge tube for dry weigth sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herasafe KS</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">51024579</td>
			<td>Laminar flow hood</td>
		</tr>
	</tbody>
</table>

spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

For the initial method tests, Synechocystis was cultivated as batch cultures in Erlenmeyer flasks on a shaker in BG11 cultivation medium20 (supplemented with 17 mM HEPES) at 25 &#176;C, under a warm white light of an intensity of 50 &#181;mol(photons)/(m2&#183;s) and with 1% CO2 in the culturing atmosphere. During the cultivation, the cultures were sampled to safe-lock tubes and centrifuged (15,000 x g at laboratory temperatu...

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Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

The overall goal of this protocol is to quantitatively determine the phycobiliproteins content in the model cyanobacterium Synechocystis using a spectrophotometric method. Phycobiliproteins are water soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria and several groups of eukaryotic algae. Since they are the main light-harvesting complexes, phycobiliproteins represent one of the crucial factors that determine algae and cyanobacteria productivity.Synechocystis is a model cyanobacterium which is used in hundreds of laboratories worldwide. Synechocystis contains two phycobiliproteins, phycocyanin and allophycocyanin. This protocol describes simple, efficient and reliable method for quantitative determination of both phycobiliproteins in this mold strain.We compared several methods, phycobiliproteins extraction and spectrophotometric quantification. The quantification method needs to be tested for every single strain since phycobiliproteins absorption spectra can vary between different strains. On the beginning of the phycobiliproteins extraction, transfer one milliliter of cyanobacterium culture to a safe-lock tube.If you decide to determine dry weight of the samples, weight the empty tubes before the culture sampling. Be sure that the culture is not sedimented. Centrifuge the cells at 15, 000 G at laboratory temperature for five minutes.Carefully discard the supernatant. Be sure to not disturb the pellet. Put the samples to freezer.For long-term storage, keep the samples at 80 degrees Celsius. This is necessary to prevent phycobiliproteins degradation. Once the samples are frozen, insert them into the freeze dryer.Once the freeze drying has started, check the temperature and pressure on the freeze dryer. The temperature has to be below 60 degrees Celsius and the pressure has to be around one hectare Pascal. Freeze dry the samples overnight.After finishing the freeze drying cycle, close the tubes as soon as possible to prevent reabsorption of water from air. Add four PCs of two milliliter glass beads to each sample tube. Homogenize the samples with the glass beads for 15 seconds.Properly homogenized sample is spread over the whole inner surface of the safe-lock tubes. After cells homogenization, add one milliliter of phosphate-buffered saline to the samples in order to extract the phycobiliproteins. Optimal pH of the PBS is around 7.4.Mix the cells with the PBS for five seconds on the homogenizer. This will secure optimal sample mixing for efficient phycobiliproteins extraction. After mixing, the samples are greenish.Keep the samples on ice for 60 minutes for the most efficient phycobiliproteins extraction. After the extraction, centrifuge the samples at 15, 000 G and four degrees Celsius for five minutes. After centrifugation, the supernatant has cyan blue color.Before the spectrophotometric measurement, calibrate the spectrophotometer using the PBS as a blank. Once the spectrophotometer is calibrated discarded PBS from one spectrophotometer cuvette and pipette the supernatant with extracted phycobiliproteins instead of the discarded buffer. For phycocyanin and allophycocyanin quantification the supernatant with extracted phycobiliproteins is measured at 615, 652 and 720 nanometers.As described in the text, there are several equations for the phycobilisomes quantification known in the literature. The equation used in this protocol was found as the best correlating phycocyanin and allophycocyanin concentrations as the pigments and dots. Critical steps of the protocol are cells homogenization and extraction, that should provide both high yield and high specificity.We tested several extraction procedures such as cells disruption by sonication, homogenization with zirconia beads, or freeze cell link. The method from this protocol provided the highest phycobilisomes yield. Since even a small contamination of the protein extract by chlorophyll can lead to the phycobiliproteins content overestimation, it is necessary to avoid any chlorophyll extraction from the cells.We detected chlorophyll in the crude extract, then we used sonication for cells disruption. With repeating sonication cycles, chlorophyll concentration increased. Homogenization time was optimized as 15 seconds.Homogenization for both five seconds and 20 seconds provided slightly lower yields. Addition of sodium or potassium chloride to the PBS buffer or to water, did not increase phycobiliproteins extraction efficiency, same as extraction in sodium acetate buffer. The phycobiliproteins extraction time was optimized for 60 minutes, since after 120 and even 240 minutes, the phycobiliproteins concentration didn't change significantly.Make sure that absorbance at 615, 652 and 720 nanometers fit into the near absorbance range of the spectrophotometer. The spectrophotometer used in this protocol show the near absorbance range between 0.1 and three. We compared several equations of spectrophotometric phycobiliproteins quantification known from the literature.The equation of Bennett and Bogorad provided the best reconstruction of both phycocyanin and allophycocyanin content in the pigment standards with known protein concentrations. The differences between the individual equations are connected with variations in phycobiliproteins extinction coefficients of the specific organism. The use of streptomycin sulfate didn't lead to reduction of chlorophyll A in samples where sonication was applied or avoided.As the representative data, we determined content of phycobiliproteins in Synechocystis cells. Phycobiliproteins content decreased under increasing light. The protocol was established to minimize time and equipment requirements for the written pigment analysis that can be performed in any standard live-science laboratory.Also, the protocol contains two other steps, such as freeze drying of the cell pellet and proteins extraction. The total labor time for the phycobilisomes quantification is no longer than two hours.

spectrophotometric-determination-phycobiliprotein-content

Research

JoVE Journal

Biochemistry

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...