<img src="/files/ftp_upload/1746/1746fig1.jpg" alt="Figure 1" /> Figure 1. The three-dimensional structure of the cystatin B complex (blue) with papain (yellow). Mutated cystatin B residues are shown in red.
1. Principles of Label-free Interaction Analysis using Biacore Systems
In a typical label-free binding experiment using a Biacore system, a biomolecule termed the 'ligand' is attached to the surface of a sensor chip. A system of flow-channels brings its binding partner, termed the 'analyte', into contact with the chip surface, where detection takes place. When the analyte binds to the ligand, the resulting change in accumulating mass at the surface is detected by surface plasmon resonance (SPR). The SPR response is proportional to the amount of binding analyte.
Since the binding is measured in real time, the kinetic association and dissociation rate constants for a specific interaction can be determined. From these constants, it is possible to calculate the affinity as the equilibrium dissociation constant. It is also possible to calculate affinity from steady state binding data. A similar methodology can also be used to determine the concentration of a protein that specifically binds to a ligand on the surface. 
2. Analyzing kinetic properties of a protein-protein interaction
In Biacore X100, kinetic analysis can be performed using single-cycle kinetics. In a single-cycle kinetics experiment, a concentration series of the analyte is injected in a single analysis cycle without regeneration of the surface in between injections. Therefore, single-cycle kinetics enables kinetic analysis when it is difficult to find suitable regeneration conditions.
Once the data for a kinetic experiment has been collected, Biacore X100 evaluation software generates the values of ka, kd, and KD by fitting the data to an interaction model.
3. Approaches to Determining Protein Concentration
Analyzing the interactions between biomolecules is important for understanding their function. Characterizing the binding of proteins to other proteins, to nucleic acid, or to small molecules is fundamental to biochemical research, and finds use in many other areas including drug discovery.
For accurate measurement of the interaction kinetics between two interacting proteins it is essential to know the concentration of specifically binding protein in the experimental sample that is used as analyte. A spectrophotometer reading of A280 or colorimetric assays such as the one employing Bradford's reagent is commonly used to determine total protein concentration. However, protein impurities can affect the result. More importantly, both active and inactive forms of the protein are included in the total protein concentration. Particularly in the case of recombinant proteins, which may be inactive due to incorrect folding, it is important to determine the percentage of specifically binding protein in the sample. 
In Biacore X100, concentration related to specific binding activity can either be determined by comparison of binding response levels to a calibration curve derived from a known standard, or by using the more recently introduced methodology Calibration-Free Concentration Analysis (CFCA). CFCA does not rely on to a standard. Therefore, CFCA is particularly useful in the case of studying mutant forms of proteins, where standards are usually not available. 
In a CFCA experiment, the initial binding rate is measured at different flow rates under conditions when diffusion of sample to the chip surface is rate-limiting. The diffusion coefficient of the analyte, the dimensions of the flow cell and the flow rates are taken into account when calculating the specific binding concentration from the initial binding rate1,2.
In a kinetics experiment, the concentration of the analyte is used in the calculation of the kinetic association rate constant and the affinity from the experimental data. CFCA and kinetic measurement in Biacore systems both rely on the same interaction properties. Therefore, using the specific binding concentration determined by Biacore analysis, rather than total protein concentration, increases the reliability of the results.
4. Using Biacore analysis to Characterize the Interaction Between Cystatin B and Papain
Mammalian cystatin B is a reversible, competitive and tight-binding protein inhibitor of papain-like cysteine proteinases, primarily cathepsin B, H, K, L and S. These proteins are mainly involved in nonselective intracellular protein degradation. Cystatins are presumed to protect cells and tissues from inappropriate proteolysis by these enzymes. They also inactivate cysteine proteinases from parasites and viruses and may participate in the defense against invasion of such infectious agents. In addition, cystatins inhibit several plant cysteine proteinases, such as papain, which is frequently used as a model enzyme in structure-function studies. The three-dimensional structure of the cystatin B complex with papain3 shows that the interaction between the two proteins is dominated by hydrophobic contacts, which, from the inhibitor side, are provided by the N- and C-terminal ends and two hairpin loops (Figure 1). In this study, we examine the importance of the C-terminal end and the second binding loop of cystatin B for binding to papain by using bovine cystatin B variants containing point mutations in areas of interaction with papain. We first determine the specific binding concentration of the four cystatin B variants used, as well as that of the wild type protein. Once the specific binding concentrations of active cystatin B are determined, the rate and affinity constants of wild type and mutant variants of cystatin B binding to papain are measured using Biacore X100.
5. Instrument and Reagents
<ol>
 <li>The cystatin B variants Cys3Ser/His75Gly, Cys3Ser/Leu73Gly, Cys3Ser/Tyr97Ala and Cys3Ser are produced as previously described4. All mutants contain an additional substitution of cysteine to serine at position 3, to prevent the formation of disulfide-linked inactive dimers of cystatin B.</li>
 <li>The papain target is also prepared as previously described5. It is S-(methylthio) papain (MMTS-papain) having a methylthio group attached to Cys25 in the active cleft, rendering the protease catalytically inactive.</li>
 <li>Biacore X100 with Biacore X100 Plus Package is used to measure and analyze the specific binding concentrations and binding kinetics. </li>
 <li>MMTS-papain is immobilized on Sensor Chip CM5 using Biacore Amine Coupling Kit.</li>
 <li>The runs are performed at 25&deg;C, and the buffer is 0.01 M Hepes at pH 7.4, 0.15 M NaCl, 0.0034 M EDTA, and 0.05% polysorbate 20.</li>
 <li>Between each variant of cystatin B, the sensor surface is regenerated with 20 mM NaOH for 30 seconds at a flow rate of 10 &mu;l/min. </li>
</ol>
6.Immobilization of the Ligand MMTS-papain
Covalent attachment of MMTS-papain for CFCA analysis
CFCA relies on measurement of binding rate under conditions where the rate is limited by diffusion of analyte molecules to the surface (mass transport limitation). This is favored by high immobilization levels of the ligand. Immobilization of MMTS-papain was set up and run using the immobilization wizard of Biacore X100 Control software.
<ol>
 <li>Activate the surface in flow cell 2 by injection of a mixture of succinimide (NHS) and carbodiimide (EDC) for 7 minutes at a flow rate of 10 &mu;l/min. Flow cell 1 is left unmodified in order to be used as a reference surface.</li>
 <li>Inject MMTS-papain at 50&mu;g/ml in sodium acetate buffer at pH 4.5 for 15 minutes at a flow rate of 5 &mu;l/min.</li>
 <li>Ethanolamine is injected for 7 minutes at a flow rate of 10 &mu;l/min to deactivate remaining active esters.</li>
</ol>
The entire coupling procedure should result in ~ 3000 RU MMTS-papain immobilized in flow cell 2.
Covalent attachment of MMTS-papain for kinetic analysis
The same procedure is used for immobilizing MMTS-papain for kinetic analysis, with the only difference being that in kinetic analysis, the immobilization level needs to be low in order to avoid the binding rate becoming limited by diffusion. Flow cell 1 is left unmodified in this step as well in order to be used as a reference surface.
<ol start="4">
 <li>After activation of the surface with the mixture of NHS and EDC as in 7.1, MMTS-papain is injected at 1&mu;g/ml for 50 seconds. After that the surface is de-activated with an injection of ethanolamine as in step 7.3.</li>
</ol>
The coupling level should be approximately 50 RU after this procedure.
7. Determining Cystatin B concentration using the CFCA assay
 <ol>
 <li>The CFCA experiment is set up according to the CFCA wizard in Biacore X100 Plus Package. Approximately 10 nM protein is injected at flow rates 10 and 100 &mu;l/min in duplicate for 48 seconds. The surface is regenerated with 20 mM NaOH between each cycle.</li>
 <li>Include blank injections for each flow rate.</li>
 <li>The protein concentration is then determined from the binding data (Figure 3) using the CFCA evaluation feature of Biacore X100 Plus Package evaluation software. <img src="/files/ftp_upload/1746/1746fig2.jpg" alt="Figure 2" /> Figure 2. Results from the CFCA analysis of wild-type cystatin B. The specific binding concentration data was extracted from the sensorgrams obtained at flow rates of 10 and 100 &mu;l/min. <img src="/files/ftp_upload/1746/1746fig3.jpg" alt="Figure 3" /> Figure 3. Concentrations of the mutants determined using A280 measurement (n=3) and CFCA (n=2). The standard errors are denoted by error bars. The molar absorption coefficients were calculated as described in (6). A large difference in the concentration values of Cys3Ser/Leu73Gly mutant determined by the two methods can be observed.</li>
 <li>When comparing the concentration determined by A280 measurement with that by CFCA, it is clear that, while in most cases the protein is fully active, for the Cys3Ser/Leu73Gly variant, the fraction of active protein is very low (Figure 3). The table below shows the activities of cystatin B variants related to the total concentrations by A280.
<table border="0">
<tbody>
 <tr>
 <td>Sample</td>
 <td>CFCA in relation to A280 (%)</td>
 </tr>
 <tr>
 <td>Wild type</td>
 <td>94</td>
 </tr>
 <tr>
 <td>Cys3Ser/His75Gly</td>
 <td>101</td>
 </tr>
 <tr>
 <td>Cys3Ser/Leu73Gly</td>
 <td>9</td>
 </tr>
 <tr>
 <td>Cys3Ser/Tyr97Ala</td>
 <td>99</td>
 </tr>
 <tr>
 <td>Cys3Ser</td>
 <td>83</td>
	</tr>
</tbody>
</table> 
</li>
</ol>
8. Measuring the Kinetics of Cystatin B binding to Papain
<ol>
	<li>Based on the CFCA measurements, prepare a two-fold concentration series ranging from 2.5 to 40 nM for each of the cystatin B variants. </li>
 <li>The kinetics experiment is set up using the kinetics wizard in Biacore X100 with the single cycle approach. The surface is not regenerated between injections, but after the end of each analysis cycle using 20 mM NaOH as regeneration solution. </li>
 <li>Set up the experiment so that each cycle containing sample is flanked by a blank cycle, where buffer is injected instead of sample. </li>
 <li>Use the kinetics evaluation feature in Biacore X100 to automatically perform blank subtractions of reference subtracted data before fitting a 1:1 interaction model. </li>
 <li>The experimental data obtained for the binding of cystatin B variants to papain is shown in Figure 4. The table below summarizes the association and dissociation rate constants and the equilibrium dissociation constants obtained in the kinetic analysis.
<table border="0">
<tbody>
<tr>
 <td>Sample</td>
 <td>ka (M-1 s-1)</td>
 <td>kd (s-1)</td>
 <td>KD (M)</td>
</tr>
<tr>
 <td>Wild type</td>
 <td>1.8 x 106</td>
 <td>0.41 x 10-3</td>
 <td>2.3 x 10-10</td>
</tr>
<tr>
 <td>Cys3Ser/His75Gly</td>
 <td>1.1 x 106</td>
 <td>1.7 x 10-3</td>
 <td>1.5 x 10-9</td>
</tr>
<tr>
 <td>Cys3Ser/Leu73Gly</td>
 <td>1.1 x 106</td>
 <td>23 x 10-3</td>
 <td>2.2 x 10-8</td>
</tr>
<tr>
 <td>Cys3Ser/Tyr97Ala</td>
 <td>1.7 x 106</td>
 <td>12 x 10-3</td>
 <td>7.1 x 10-9</td>
</tr>
<tr>
 <td>Cys3Ser</td>
 <td>0.9 x 106</td>
 <td>0.53 x 10-3</td>
 <td>5.8 x 10-10</td>
</tr>
</tbody>
</table>
 <img src="/files/ftp_upload/1746/1746fig4.jpg" alt="Figure 4" /> Figure 4. Kinetic profiles for cystatin B variants binding to MMTS-papain determined using single cycle kinetics. Sensorgrams show blank- and reference subtracted data with kinetic fit for 1:1 interaction model overlaid in black.</li>
 <li>While the association rates of the mutants are comparable to that of the wild type protein (Figure 5, upper panel), the dissociation rate constants of the mutants can be higher by close to two orders of magnitude, with a corresponding increase in the equilibrium dissociation constant (Figure 5, lower panel). <img src="/files/ftp_upload/1746/1746fig5.jpg" alt="Figure 5" /> Figure 5. The calculations of the rate and affinity constants were based on the concentrations obtained from CFCA. The changes of ka, kd and KD, associated with the mutations are depicted in the graphs.</li>
	<li>Since sample concentration is a parameter in the calculation of the association rate constant from the experimental data, it is important that it is correct. Using the concentration based on the A280 measurement instead of CFCA would lead to the conclusion that Leu73 is important for the association rate, where replacement seems to result in about 10 times slower association than the other cystatin B variants. However, the specific binding concentration measured by CFCA is only about 10% of the total protein in this case. When this is taken this into account it becomes clear that the association rate for Leu73 is similar to that of the other variants. Therefore, CFCA allows for correct assessment of ka and KD thus making possible the appropriate interpretation of the interaction mechanism.</li>
</ol>

<ol>
	<li>Christensen, L. L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+protein+concentration+determination+using+biosensor+technology+under+conditions+of+partial+mass+transport+limitation.">Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.</a> Anal. Biochem. 249, 153-164 (1997).</li><li>Sigmundsson, K., M&#226;sson, G., Rice, R., Beauchemin, N., &#214;brink, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+active+concentrations+and+association+and+dissociation+rate+constants+of+interacting+biomolecules:+an+analytical+solution+to+the+theory+for+kinetic+and+mass+transport+limitations+in+biosensor+technology+and+its+experimental+verification.">Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification.</a> Biochemistry. 41 (26), 8263-8276 (2002).</li><li>Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., Turk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+refined+2.4+A+X-ray+crystal+structure+of+recombinant+human+stefin+B+in+complex+with+the+cysteine+proteinase+papain:+a+novel+type+of+proteinase+inhibitor+interaction.">The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.</a> EMBO J. 9 (6), 1939-1947 (1990).</li><li>Pol, E., Bj&#246;rk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+second+binding+loop+and+the+C-terminal+end+of+cystatin+B+(Stefin+B)+for+inhibition+of+cysteine+proteinases.">Importance of the second binding loop and the C-terminal end of cystatin B (Stefin B) for inhibition of cysteine proteinases.</a> Biochemistry. 38 (32), 10519-10526 (1999).</li><li>Bj&#246;rk, I., Pol, E., Raub-Segall, E., Abrahamson, M., Rowan, A. D., Mort, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+changes+in+the+association+and+dissociation+rate+constants+for+binding+of+cystatins+to+target+proteinases+occurring+on+N-terminal+truncation+of+the+inhibitors+indicate+that+the+interaction+mechanism+varies+with+different+enzymes.">Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes.</a> Biochem. J. 299, 219-225 (1994).</li><li>Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., Gray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+the+molar+absorption+coefficient+of+a+protein.">How to measure and predict the molar absorption coefficient of a protein.</a> ProteinScience. 4 (11), 2411-2423 (1995).</li></ol>

The author is employed by GE Healthcare that produces reagents and instruments used in this article.

In this work, four mutants and wild type cystatin B were produced in order to assess the importance of the second binding loop and C-terminal ends for the interaction between cystatin B and papain. This study demonstrated the advantage and ease of using Biacore X100 to determine specific binding concentration and to analyze the kinetics of protein-protein interactions in order to understand structure-function relationships. We showed that measuring total protein concentration does not reveal the protein variants that have reduced binding activity, which in this case introduces a significant measurement error in the determination of binding affinity and kinetics. The specific binding concentrations of the cystatin B variants were determined using CFCA with Biacore X100. Using concentration measurements by CFCA as input to the kinetic analysis resulted in reliable rate and affinity constants, thus allowing the right interpretation of the interaction mechanism.
The decreased affinities of the mutants were almost exclusively due to an increased kd-value, whereas ka was affected only slightly. This behavior indicates that both the second binding loop region and the C-terminal end are not important for the binding rate of inhibitor to papain. Instead, they contribute to binding affinity primarily by keeping the inhibitor attached to the enzyme once the complex has been formed.

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<td>Biacore&trade; X100 System</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1100-73</td>
<td><a href="http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html">http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html</a></td>
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<td>Biacore&trade; X100 Plus Package</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
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the importance of correct protein concentration for kinetics and affinity determination in structure-function analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

Watch this Scientific Journal Video about The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis at JoVE.com

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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20.5K Views.  GE Healthcare Bio-Sciences AB. نحن نطبق التسمية خالية من تحليل التفاعل البروتين باستخدام X100 Biacore للهيكل وظيفة تحليل الربط العديد من المسوخ B cystatin إلى غراء من خلال توصيف الحركية. المعايرة خالية من التحليل تركيز (CFCA) يقيس تركيز البروتين مع النشاط ملزم الاحتفاظ دون الحاجة إلى منحنى القياسية. نظهر...

Video: The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...