This work has been supported by Wellcome Trust grant 105278/Z/14/2 to R.N. The Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust Core Award 203852/Z/16/2. Work in the C.S. group is supported by Cancer Research UK (C20724/A14414), and the European Research Council under the European Union's Horizon 2020 Research and Innovation Programme Grant 647278.

1. FKBP12 F36V -mVenus Purification
<ol>
	<li>Transform (DE3) pLysS cells with pET22b vector containing monomerized human FKBP12F36V12 and N-terminal His6 and mVenus tags (vector available on request). Plate cells onto LB agar supplemented with 50 &#181;g/mL Ampicillin and 34 &#181;g/mL Chloramphenicol.</li>
	<li>Transfer transformed colonies into 100 mL LB starter culture and grow for 16 - 20 hours at 37 &#176;C with shaking.</li>
	<li>Dilute dense starter culture (OD600 &#62;1) 1:100 in LB medium (2 x 500 mL batches) and grow for 2 - 3 hours to OD600 = 0.6 - 0.8 (37 &#176;C, 200 rpm ).</li>
	<li>Cool cultures on ice. Induce with 250 &#181;M IPTG and grow for 16 - 20 hours at 21 &#176;C, 200 rpm.</li>
	<li>Harvest cells by centrifugation at 2,000 x g for 20 minutes.</li>
	<li>Resuspend pellet in 40 mL of IMAC buffer A (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 3 mM imidazole, 1 mM &#946;-mercaptoethanol) supplemented with EDTA-free protease inhibitors (1 tablet per cell pellet).</li>
	<li>Sonicate cells (500 Watts, 20 kHz, 40% amplitude, 9 s on, 11 s off for 15 min) on ice and harvest soluble material by centrifugation at 20,000 x g.</li>
	<li>Transfer soluble lysate to a conical flask and add 2 mL of resin (see Table of Materials). Incubate for 1 hour with 105 rpm rotation 
	NOTE: Nickel sepharose may also be used for this IMAC step.</li>
	<li>Harvest resin and wash with 250 mL of IMAC buffer A followed by 500 mL of IMAC buffer B (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 7 mM imidazole, 1 mM &#946;-mercaptoethanol). 
	NOTE: Increase to 50 mM imidazole if using Nickel sepharose resin.</li>
	<li>Elute His6-tagged protein using IMAC buffer C (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 300 mM imidazole, 1 mM &#946;-mercaptoethanol).</li>
	<li>Inject onto a size exclusion column (see Table of Materials) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. FKBP12F36V has its peak elution at 87.71 mL on the column we used.</li>
	<li>Assess purity via SDS-PAGE and pool and concentrate as required.</li>
</ol>
2. Preparation of Multiwell Plate Array
<ol>
	<li>Thaw the purified FKBP12F36V (or labeled protein of interest) from -80 &#176;C.</li>
	<li>Prepare a solution of 100 nM purified FKBP12F36V (medium, 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT). Sonicate and centrifuge (quick spin of 13,000 rpm) to prevent the formation of aggregates.</li>
	<li>Pipette 100 - 200 &#181;L into an 8 well observation chamber with a glass bottom.</li>
	<li>Add the BB dimerizer to final concentrations of 10, 20, 40, 80, 100, 150, 300, and 500 nM12,13.</li>
	<li>As a reference, prepare a solution of 100 nM of mVenus alone to evaluate potential aggregation and precipitation effects and recover a brightness value for the monomer with the same acquisition settings.</li>
</ol>
3. Calibration-free Confocal Acquisition
<ol>
	<li>Start the confocal system (Figure 1). Any light scanning microscope confocal system equipped with digital detectors or well-characterized analog detectors8, and capable of keeping a constant dwell time for every pixel acquired would work.</li>
	<li>Set the excitation beam path:
	<ol>
		<li>Turn on the 514 nm laser and set it at 20 - 100 nW Power at the exit of the objective (for FKBP12F36V-mVenus).</li>
		<li>Select the 63X1.4NA objective or a collar correction water immersion objective designed for FCS.</li>
		<li>Turn on one HyD, APD or calibrated PMT detector. Detectors capable of photon-counting are preferable, as in this case, calculation of S, offset and &#963;02&#160;are unnecessary.</li>
		<li>Select the emission window from 520 - 560 nm</li>
		<li>Set the pinhole at 1 Airy unit for the corresponding emission ~545 nm.</li>
		<li>Set the acquisition mode at 16 x 16 pixels</li>
		<li>Set the pixel dwell time tdwell such that it satisfies tframe &#62;&#62; TD &#62;&#62; tdwell, where D is the residence time of the diffusing protein and tframe is the frame rate. This corresponded to setting the dwell time to ~13 &#181;s. 
		Note: Some commercial manufacturers had scanners that were not keeping the dwell-time per pixel constant. This constancy is crucial for the method to work.</li>
		<li>Set the pixel size at ~120 nm.</li>
		<li>Select the xyt acquisition mode and select the number of frames to be acquired per acquisition and well (for example 5,000).</li>
		<li>If the system is equipped with high-throughput mode, introduce the coordinates of each well and the number of acquisitions per well to automate the process. 
		Note: Be careful to ensure the presence of a water dispenser for if using an immersion objective.</li>
		<li>If the system is equipped with a perfusion system, load the BB solution and program the perfusion to start right-after the 5000th frame to evaluate the kinetics of dimerization while acquiring e.g., 10,000 images.</li>
	</ol>
	</li>
	<li>Add a drop of oil into the oil immersion objective / water if utilizing a collar correction water immersion objective designed for FCS.</li>
	<li>Mount the 8 well observation chamber into the stage.</li>
	<li>Select the correct well and focus on the solution. 
	Note: IMPORTANT: Avoid focusing close to the bottom glass to avoid reflection and scattering. When focusing deeper into the solution, disconnect the automated focus option.</li>
	<li>Start the acquisition and save the resulting stack of images in TIFF format.</li>
</ol>
4. Detrend and Brightness Analysis using the R Package nandb
<ol>
	<li>As a preprocessing quality check, use ImageJ14 to take a look at the images and recover the intensity profile, as shown in Figure 2a. This is useful to determine whether or not too much photobleaching has occurred. If there is too much bleaching, the data is not suitable for further analysis. 
	Note: ImageJ can also be useful to convert images to TIFF from commercial formats. The nandb software described below can only work with TIFF files.</li>
	<li>Download and install R and RStudio15,16. It is best to download and install R first, then RStudio. 
	&#8203;NOTE: What follows is a description of how to use the nandb R package. Knowledge of the R language is not required to use nandb, however it will make life easier.</li>
	<li>Install the nandb package.
	<ol>
		<li>Open RStudio and in the console, type install.packages(&#34;nandb&#34;) and wait for the installation.</li>
	</ol>
	</li>
	<li>Get to know nandb
	<ol>
		<li>Review the manual17.</li>
		<li>Review the built-in RStudio help for various functions. The most likely function to be used will be using will be brightness(). View the help file for this function by typing ? brightness() at the console.</li>
	</ol>
	</li>
	<li>Calculate brightness
	<ol>
		<li>Say one has an image file on the desktop called img001.tif (note that `nandb` only works with TIFF files). One can calculate the brightness of that image: 
		&#8203;b &#60;- brightness(&#34;~/Desktop/img001.tif&#34;, tau = &#34;auto&#34;)
		<ol>
			<li>This assigns the brightness of the image to the variable b in R. The tau = &#34;auto&#34; ensures that the image is correctly detrended prior to brightness calculation. The most common thing to do from here is to calculate the mean or median brightness of the image. One can do this by typing mean(b) or median(b). One can also write the brightness image to the desktop using 
			ijtiff::write_tif(b, &#34;~/Desktop/whatever_img_name&#34;)</li>
		</ol>
		</li>
		<li>Say one has folder full of images images_folder on the desktop and one needs to calculate the brightnesses of these images and write the brightness images as TIFF files. Then see ?brightness_folder(). This function processes a whole folder all at once: 
		brightness_folder(&#34;~/Desktop/images_folder&#34;, tau = &#34;auto&#34;) 
		This is particularly good for those who have a software they prefer to R, because all of the files are processed in one single command, and then one can go on working with the output brightness TIFF images in their chosen software, be it ImageJ14, Python or something else.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Voith von Voithenberg, L., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+pair+forster+resonance+energy+transfer:+A+versatile+tool+to+investigate+protein+conformational+dynamics.">Single pair forster resonance energy transfer: A versatile tool to investigate protein conformational dynamics.</a> BioEssays. 40, (2018).</li><li>Padilla-Parra, S., Auduge, N., Coppey-Moisan, M., Tramier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+by+fast+acquisition+time+domain+FLIM+at+high+spatial+resolution+in+living+cells.">Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells.</a> Biophysical Journal. 95, 2976-2988 (2008).</li><li>Padilla-Parra, S., Auduge, N., Coppey-Moisan, M., Tramier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-color+fluorescence+lifetime+correlation+spectroscopy+to+quantify+protein-protein+interactions+in+live+cell.">Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell.</a> Microscopy Research and Technique. 74, 788-793 (2011).</li><li>Muller, J. D., Chen, Y., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy.">Fluorescence correlation spectroscopy.</a> Methods in Enzymology. 361, 69-92 (2003).</li><li>Tramier, M., Coppey-Moisan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+anisotropy+imaging+microscopy+for+homo-FRET+in+living+cells.">Fluorescence anisotropy imaging microscopy for homo-FRET in living cells.</a> Methods in Cell Biology. 85, 395-414 (2008).</li><li>Digman, M. A., Dalal, R., Horwitz, A. F., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+number+of+molecules+and+brightness+in+the+laser+scanning+microscope.">Mapping the number of molecules and brightness in the laser scanning microscope.</a> Biophysical Journal. 94, 2320-2332 (2008).</li><li>Nolan, R., Iliopoulou, M., Alvarez, L., Padilla-Parra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein+aggregation+and+interaction+in+live+cells:+A+guide+to+number+and+brightness.">Detecting protein aggregation and interaction in live cells: A guide to number and brightness.</a> Methods. , (2017).</li><li>Dalal, R. B., Digman, M. A., Horwitz, A. F., Vetri, V., Gratton, E. <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+particle+number+and+brightness+using+a+laser+scanning+confocal+microscope+operating+in+the+analog+mode.">Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode.</a> Microscopy Research and Technique. 71, 69-81 (2008).</li><li>Nolan, R., 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=nandb-number+and+brightness+in+R+with+a+novel+automatic+detrending+algorithm.">nandb-number and brightness in R with a novel automatic detrending algorithm.</a> Bioinformatics. , (2017).</li><li>Hur, K. H., 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=Quantitative+Measurement+of+Brightness+from+Living+Cells+in+the+Presence+of+Photodepletion.">Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion.</a> PLoS One. 9, (2014).</li><li>Amara, J. F., 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=A+versatile+synthetic+dimerizer+for+the+regulation+of+protein-protein+interactions.">A versatile synthetic dimerizer for the regulation of protein-protein interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 10618-10623 (1997).</li><li>Clackson, T., 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=Redesigning+an+FKBP-ligand+interface+to+generate+chemical+dimerizers+with+novel+specificity.">Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 10437-10442 (1998).</li><li>Rollins, C. T., 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=A+ligand-reversible+dimerization+system+for+controlling+protein-protein+interactions.">A ligand-reversible dimerization system for controlling protein-protein interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 7096-7101 (2000).</li><li>Schindelin, J., Rueden, C. T., Hiner, M. C., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageJ+ecosystem:+An+open+platform+for+biomedical+image+analysis.">The ImageJ ecosystem: An open platform for biomedical image analysis.</a> Molecular Reproduction and Development. 82, 518-529 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+Language+and+Environment+for+Statistical+Computing.">R: A Language and Environment for Statistical Computing.</a> R Core. , (2017).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RStudio:+Integrated+Development+Environment+for+R.">RStudio: Integrated Development Environment for R.</a> R Team. , (2016).</li><li>. nandb R package Available from: <a target="_blank" href="https://CRAN.R-project.org/package=nandb">https://CRAN.R-project.org/package=nandb</a> (2017)</li><li>Jung, G., Wiehler, J., Zumbusch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photophysics+of+green+fluorescent+protein:+influence+of+the+key+amino+acids+at+positions+65,+203,+and+222.">The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222.</a> Biophysical Journal. 88, 1932-1947 (2005).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nature Methods. 2, 905-909 (2005).</li><li>Butkevich, A. N., 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=Hydroxylated+fluorescent+dyes+for+live-cell+labeling:+synthesis,+spectra+and+super-resolution+STED.">Hydroxylated fluorescent dyes for live-cell labeling: synthesis, spectra and super-resolution STED.</a> Chemistry: A European Journal. 23, 12114-12119 (2017).</li></ol>

The authors have nothing to disclose.

N&#38;B is a technique to detect multimerization using commercial light scanning confocal microscopes equipped with digital detectors. This approach is quite attractive compared to single point FCS, FCCS, and smFRET because it is calibration free and the brightness calculation is straightforward and concentration independent6. It is of major importance, however, to correct for bleaching and long-term intensity fluctuations before performing brightness calculations9; a sligh...

Protein-protein Interactions In Vitro
Traditionally, crystallography and nuclear magnetic resonance experiments combined with cryo-electron microscopy (cryoEM) are the technologies chosen to accurately describe the three-dimensional architecture of proteins and to infer their function by scrutinizing their high resolution structural details. Proteins, however, are not static structures and can undergo a variety of conformational changes and vibrations in time and space. This is why structural information from crystallographic or CryoEM data needs to be complemented with other techniques (e.g., molecular dynamics simulations and single molecule techniques): the function of a protein is related to its conformational changes and interactions, and this information is not present in a static structure. In order to probe for intra-molecular dynamics, techniques based on single molecule Forster Resonance Energy Transfer (smFRET) are very effective1. These approaches are able to assess different subpopulations of molecules in complex media. This is very important, as these changes are rapid and occur during the acquisition of the data (i.e.,&#160;nanosecond to second range).
Two main approaches are commonly employed to detect and quantify these changes: proteins in solution and surface-immobilization. For the detection of inter-molecular interactions and in particular, the process of dimerization induced by ligands, smFRET is not always the best tool. Indeed, FRET depends not only on the distance (&#8776;10 nm) but also on the orientation of the two dipoles (donor and acceptor, &#967;2) and the overlap of the donor emission with the acceptor&#39;s absorption spectra2, but perhaps this last condition is less important provided that the experimentalist can chose the right FRET couple. A particular disadvantage of smFRET for probing homo-dimerization comes from the labeling of the protein of interest: for hetero smFRET, dimerization can only be detected up to 50% (i.e.,&#160;hetero-FRET will only be able to detect donor-acceptor and acceptor-donor homo-dimers but not donor-donor or acceptor-acceptor, which is the other 50% of the dimers). The use of fluorescence correlation spectroscopy (FCS) and derivatives (FCCS, etc.3) to ascertain protein diffusion constants and binding constants in vitro is another alternative. These approaches are not able to fully quantify homo-dimerization either, as in FCS one measures concentration and diffusion, and the radius and diffusion coefficient of a diffusing particle are very poorly dependent on the molecular weight; for example a 10-fold increase in the molecular weight will only imply a 2.15 fold change in the diffusion coefficient4. In the case of two-color FCS or FCCS, only 50% of homo-dimers will be seen for the same reason as above. The most practical and quantitative approaches to detect homo-dimerization in vitro and in vivo are homo-FRET5 and number and brightness (N&#38;B)6. Given the fact that homo-FRET requires specific instrumentation recovery of the anisotropy value (i.e.,&#160;optical elements/analyzers to recover the parallel and perpendicular polarization), N&#38;B is presented here as a favorable technique to detect protein homo-dimerization and aggregation. It can be employed both in vitro and in vivo with a commercial set-up.
Number and Brightness
N&#38;B has been recently reviewed7. That review focused on the application of the technique in live cells. It is worthwhile to reproduce the mathematical formalism here as these equations will be applied to the data collected in vitro. First, it is necessary to define some terms and mathematical quantities:
<ul>
	<li>An entity is a set of molecules which are bound together.</li>
	<li>The brightness &#949; of an entity is the number of photons it emits per unit time (per frame).</li>
	<li>n is the number of entities present.</li>
	<li>For a given pixel over the course of an image series, &#60;I&#62; is its mean intensity and &#963;2 is the variance in its intensity.</li>
</ul>
Then, with photon-counting detectors and assuming mobile entities and no background,
<img alt="Equation 1" src="/files/ftp_upload/58157/58157eq1.jpg" /> 
<img alt="Equation 2" src="/files/ftp_upload/58157/58157eq2.jpg" />
where N is the apparent number and B is the apparent brightness. This results in
<img alt="Equation 3" src="/files/ftp_upload/58157/58157eq3.jpg" /> 
<img alt="Equation 4" src="/files/ftp_upload/58157/58157eq4.jpg" />
Dalal et al.8 showed that with analog equipment, one needs three correction terms: the S factor, the background offset, and the readout noise &#963;02. Then, again assuming mobile entities,
<img alt="Equation 5" src="/files/ftp_upload/58157/58157eq5.jpg" /> 
<img alt="Equation 6" src="/files/ftp_upload/58157/58157eq6.jpg" />
giving
<img alt="Equation 7" src="/files/ftp_upload/58157/58157eq7.jpg" /> 
<img alt="Equation 8" src="/files/ftp_upload/58157/58157eq8.jpg" />
Note that the above equation for is different that given in Dalal et al.8 and a subsequent review.7 In Dalal et al. the S in the denominator was omitted due to a typo and this error was reproduced in the review. The equation above is the correct one. Instructions for measuring S, offset and &#963;02&#160;- together with an explanation of their meaning - are given by Dalal et al.8
The brightness&#160;&#949; is proportional to the oligomeric state of the diffusing entities:&#160;&#949; will be twice as big for dimers as it is for monomers, three times as big for trimers as it is for monomers, twice as big for hexamers as it is for trimers and so on. In this way, measuring the brightness &#949;, one can quantify any type of multimerization.
If there are a mixture of oligomeric states present, number and brightness is not capable of recovering the individual oligomeric states present. This is a limitation of the technique.
Detrend algorithm and nandb software
The importance of correcting for photobleaching has been previously stressed9. Photobleaching inevitably occurs during light microscopy experiments in time-lapse mode; both in live cells and in vitro. Many approaches have been described in the literature to correct for bleaching7. The exponential filtering technique with automatic choice of detrending parameter T&#160;is the current best. It is integrated into the free, open source software nandb9. Indeed, software that requires the user to manually choose their detrending parameter can lead to incorrect results because this parameter choice will likely be arbitrary and incorrect. The automatic algorithm inspects the data and determines the appropriate parameter for it, without the need for user intervention9. Even with the best choice of smoothing parameter, detrending has its limitations and works well only with photobleaching percentages lower than 25%, as shown with simulations9. Interestingly, when using the automatic detrending routine, its accuracy is such that one can work with low brightness values (even B &#60;1.01), and hence low intensities, and still be precise enough to quantify homo-dimerization.
Photobleaching also causes another problem: the presence of photobleached fluorophores in a multimer complex. This makes e.g., a trimer appear like a dimer when one of the three units in the trimer is non-fluorescent. Hur and Mueller10 showed how to correct for this and this correction was also stressed in a subsequent review7. The nandb software includes this correction9.
The FKBP12F36V system
FKBP12F36V is a protein which does not naturally oligomerize but is known to dimerize upon the addition of the AP20187 drug (colloquially known as the BB dimerizing ligand)11,12. This makes it an ideal test case for number and brightness: with labelled FKBP12F36V, a doubling of oligomeric state should be observed upon addition of BB.

<table><tbody><tr><td>RosettaTM (DE3) pLysS cells</td><td>Novagen</td><td>70956-3</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>PubChem Substance ID 329824407</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma Aldrich</td><td>PubChem Substance ID: 24892250</td><td></td></tr><tr><td>LB starter culture</td><td>QIAGEN</td><td></td><td></td></tr><tr><td>LB medium</td><td>QIAGEN</td><td>https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif</td><td></td></tr><tr><td>IPTG</td><td>Sigma Aldrich</td><td>PubChem Substance ID 329815691</td><td></td></tr><tr><td>IMAC buffer</td><td>Medicago</td><td>09-1010-10</td><td></td></tr><tr><td>EDTA-free protease inhibitors&amp;nbsp;</td><td>Sigma Aldrich</td><td>11873580001</td><td></td></tr><tr><td>TALON resin</td><td>Clonetech</td><td></td><td></td></tr><tr><td>Nickel sepharose</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>S200 16/60 column</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Glass bottom 8 well observation dish</td><td>Ibidi</td><td>80827</td><td></td></tr></tbody></table>

calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Detrending and monomeric brightness
Once the data has been acquired, one can start the brightness calculations to determine the oligomeric state of the protein of interest in the solution. Even if the effect of bleaching in solution may not be as drastic as it can be in vivo, the intensity trace will still probably not have stationary mean, possibly due to photophysical effects related to the fluorophor...

Watch this Scientific Journal Video about Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software a

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

calibration-free-vitro-quantification-protein-homo-oligomerization

Research

JoVE Journal

Biochemistry

7.2K Views.  University of Oxford. Dit protocol beschrijft een kalibratie-vrije aanpak voor het kwantificeren van eiwit homo-oligomerisatie in vitro op basis van fluctuatiemarges Fluorescentiespectroscopie met behulp van commerciële licht scanning microscopie. De juiste overname-instellingen en de analysemethoden worden weergegeven.

Video: Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Biology

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Photometric Protein Determination

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown.
This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye.
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	Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of all time. This video will show the concepts behind some of the most common photometric protein determination techniques, how they are performed, and how the gathered data is analyzed.
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	Photometric protein determination is based on the relationship between concentration and light absorbency. This is known as the Beer-Lambert Law, which states that the concentration of a light-absorbing species is proportional to its absorbance.
	This principle underlies all photometric protein determination methods. 
	For direct absorption analysis, the absorbance values of unaltered protein samples are measured. Because of their aromatic side chains, tryptophan and tyrosine residues give the highest absorbance readings at a wavelength of 280 nm.
	However, these amino acids-which are two of the least frequently found in proteins-are present in different amounts in every protein, so each determination is unique. To overcome this limitation, more complex assays-that are not dependent on these amino acids-were developed.
	One example is the Bradford Assay, where colored dye is added to the sample. The dye, known as Coomassie Blue, responds proportionally-the more protein present, the more binding events with the dye.
	Then, protein concentration is determined by measuring the absorbance of the bound Coomassie Blue dye, which absorbs light at 594 nm. However, the Bradford assay is linear over a short range of concentrations, so dilutions are often required before analysis.
	The Lowry Method combines the Biuret reagent, an alkaline solution of copper ions that react with peptide bonds, and the Folin-Cioc&#226;lteu reagent, which oxidizes aromatic protein residues. The resulting color change of the sample is proportional to the protein concentration.
	The absorbance of the reduced Folin reagent can be determined at 750 nm. Like direct absorption, each protein has a unique response, and must be calibrated for the protein of interest. Now that we've reviewed the basic principles behind some of the most common assays, let's look at how direct absorption and the Bradford assay are performed.
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	To begin a direct absorption analysis, the spectrophotometer is calibrated with a blank to determine zero absorbance. Standard solutions are prepared for use in creating the calibration curve. Then, an aliquot of the first standard is added to a cuvette, and placed into the spectrophotometer.
	The absorbance value at 280 nm is then recorded. This process is repeated for each standard, using a clean cuvette for each run. Once complete, a calibration curve is created by plotting the absorbance versus concentration. The slope of this line is the molar attenuation coefficient, which relates absorbance to concentration.
	Next, the unknown sample is added to a cuvette, and the absorbance value is recorded. As the data analysis for the different photometric determination methods is similar, we will cover that after we look at the Bradford assay.
	Here, the Bradford protein assay is performed with a BSA standard on a 96-well plate. To begin, BSA stock solutions are prepared.
	The unknown solutions are diluted with deionized water to ensure that the concentrations are within the assay's range. Depending on the kit, the Coomassie dye may also require dilution. Then, the calibration curve is set up by adding the BSA standards to the 96-well plate.
	Deionized water is added to reach the needed concentration to generate a standard curve. The unknown sample should be added to the plate in triplicates to ensure an accurate measurement is taken. Coomassie dye is next added to each well, mixing with the pipette.
	Deionized water is added to an empty well as a blank, to measure the absorbance. After waiting 5 min for the dye to bind, the absorbance is measured in a plate-reader at 590 nm.
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	Now that we've performed a few assays, let's look at how to analyze the data. Each photometric protein determination method is based on the Beer-Lambert Law.
	The measured absorbance of the standards is used to create a calibration curve, which is then used to determine the concentration of unknown samples. This curve can be manually plotted, though newer spectrophotometric tools will create the calibration curve once all standards have been measured. These systems will also calculate protein concentration as unknown samples are analyzed.
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	Now that we've reviewed how to analyze photometric protein determination data, let's look at some of the ways these procedures are utilized.
	The principles of photometric protein determination can also be used to directly measure nucleic acid concentration. The nanodrop spectrophotometer accepts samples of very small volume onto an optically active pedestal. The absorbance is then measured, and the system automatically determines the nucleic acid concentration. Because proteins and other sources can interfere with measurements, sample purity is determined by analyzing the 260 to 280 nm and 260 to 230 nm absorbance ratios. Pure nucleic acids typically yield ratios of approximately 1.8 and approximately 2.0 for DNA and RNA, respectively.
	Photometric protein determination can also be used in the production of biomimetic materials, which are inspired from nature to elicit specific cellular responses. Recombinant adhesins are bound to polystyrene beads to simulate bacterial attachment to host cells. The Bradford assay is used to determine the coupling efficiency of the recombinant adhesion to the beads in the production of the biomimetic material.
	Alternative photometric protein assays can be used in the detection and characterization of protein antimicrobials. Remazol brilliant blue R dye is covalently bonded to heat-killed bacteria. The protein antimicrobial is incubated in the dyed solution. Then, the sample is centrifuged, and the absorbance of the supernatant at 595 nm is measured using a microplate spectrophotometer. Increased absorbance, by the soluble dye released into the supernatant from the labeled bacteria, is a quantitative measurement of enzymatic activity.
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	You've just watched JoVE's video on photometric protein determination. This video described the underlying principles of photometric determination, went over general procedures for some common assays, and covered some new advances in techniques. Thanks for watching!
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Photometric Protein Determination: Bradford Assay

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

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