Name: contrast-matching detergent in small-angle neutron scattering experiments for membrane protein structural analysis and ab initio modeling
Uploaded: 2018-10-21T12:30:00
Description: Watch this Scientific Journal Video about Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling at JoVE.com

The Office of Biological and Environmental Research supported research at ORNL&#39;s Center for Structural Molecular Biology (CSMB) and Bio-SANS using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. Structural work on membrane proteins in the Lieberman lab has been supported by NIH (DK091357, GM095638) and NSF (0845445).

1. Prepare and Submit a Neutron Facility Beam Time and Instrument Proposal
<ol>
	<li>Consult online resources for identifying neutron scattering facilities that provide general user neutron beam time access, such as Oak Ridge National Laboratory (ORNL). A map of neutron facilities and information about neutron research worldwide is available online.46&#160;Be aware that these facilities typically have regular calls for proposals; this determines when the next beam time will be available. Neutron...

Structural biology researchers take advantage of complementary structural techniques like solution scattering to obtain biochemical and structural details (such as overall size and shape) from biomolecules in solution. SANS is a particularly attractive technique for determining low resolution structures of membrane proteins, a core focus of modern structural biology and biochemistry. SANS requires quantities of purified proteins comparable to those of crystallographic trials (1 mg/sample). The recently expanding commerci...

Membrane proteins are encoded by an estimated 30% of all genes1 and represent a strong majority of targets for modern medicinal drugs.2 These proteins perform a wide array of vital cellular functions,3&#160;but despite their abundance and importance &#8212;&#160;only represent about 1% of total structures deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank.4 Due to their partially hydrophobic nature, structural determination of membrane-bound proteins has been exceedingly challenging.5,6,7
As many biophysical techniques require monodisperse particles in solution for measurement, isolating membrane proteins from native membranes and stabilizing these proteins in a soluble mimic of the native membranes has been an active area of research in recent decades.8,9,10 These investigations have led to the development of many novel amphiphilic assemblies to solubilize membrane proteins, such as nanodiscs,11,12,13&#160;bicelles,14,15&#160;and amphipols.16,17&#160;However, the use of detergent micelles remains one of the most common and straightforward approaches for satisfying the solubility requirements of a given protein.18,19,20,21,22,23,24,25&#160;Unfortunately, no single detergent or magic mixture of detergents currently exists that satisfies all membrane proteins; thus, these conditions must be empirically screened for the unique requirements of each protein.26,27
Detergents self-assemble in solution above their critical micelle concentration to form aggregate structures called micelles. Micelles are composed of many detergent monomers (typically ranging from 20-200) with hydrophobic alkyl chains forming a micelle core and hydrophilic head groups arranged in a micelle shell layer facing the aqueous solvent. The behavior of detergents and micelle formation has been classically described by Charles Tanford in The Hydrophobic Effect,28&#160;and sizes and shapes of micelles from commonly used detergents in membrane protein studies have been characterized using small-angle scattering.29,30&#160;Detergent organization about membrane proteins has also been studied, and the formation of protein-detergent complexes (PDCs) is expected with detergent molecules surrounding the protein in an arrangement that resembles the neat detergent micelles.31
One added advantage in using detergents is that the resulting micelle properties can be manipulated by incorporating other detergents. Many detergents exhibit ideal mixing, and select properties of mixed micelles may even be predicted from the components and ratio of mixing.22&#160;However, the presence of detergent can still present challenges for biophysical characterizations by contributing to the overall signal. For example, with X-ray and light scattering techniques, signal from detergent in the PDC is practically indistinguishable from protein.32&#160;Investigations with single-particle cryo-electron microscopy (cryo-EM) typically rely on trapped (frozen) particles; structural details of the protein are still obscured by certain detergents or a high concentration of detergent which adds to the background.33&#160;Alternate approaches toward interpreting the full PDC structure (including the detergent) have been made through computational methods which seek to reconstruct the detergent around a given membrane protein.34
For the case of neutron scattering, the core-shell arrangement of detergent in the micelle produces a form factor which contributes to the observed scattering. Fortunately, solution components can be altered such that they do not contribute to the net observed scattering. This &#34;contrast matching&#34; process is achieved by substituting deuterium for hydrogen to achieve a scattering length density that matches that of the background (buffer). A judicious choice of detergent (with available deuterated counterparts) and their ratio of mixing must be considered. For detergent micelles, this substitution can be performed using a detergent with the same head group but having a deuterated alkyl chain (d-tail instead of h-tail). Since the detergents are well-mixed,35&#160;their aggregates will have a scattering length density that is the mole-fraction weighted average of the two components (h-tails and d-tails). When this average contrast is consistent with that of the head group, the uniform aggregate structures can be fully matched to remove all contributions to observed scattering.
We present here a protocol to manipulate the neutron contrast of detergent micelles by incorporating chemically identical detergent molecules with deuterium-labeled alkyl chains.19,36,37&#160;This permits complete simultaneous contrast matching of micelle core and shell, which is a unique capability of neutron scattering.35,38&#160;With this significantly refined level of detail, contrast matching can enable otherwise unfeasible studies of membrane protein structures. Additionally, this contrast-matching approach could be extended to other systems involving detergent, such as polymer exchange reactions39 and oil-water dispersants,40&#160;or even other solubilizing agents, such as bicelles,41&#160;nanodiscs,42&#160;or block copolymers.43&#160;A similar approach as outlined in this manuscript, but employing a single detergent species with partial deuterium substitutions on the alkyl chain and/or head group, was recently published.37&#160;While this can be expected to improve the random distribution of hydrogen and deuterium throughout the detergent compared to the approach presented here, the limited number of available positions on the detergent for substitution and two-step detergent synthesis required poses additional challenges for consideration.
Steps 1 and 2 of the protocol detailed below often overlap since initial experiment planning must be done to submit a quality proposal. However, proposal submission is considered here as the first step to emphasize that this process should be started well in advance of a neutron experiment. It should also be noted that a prerequisite step, which should be demonstrated by the proposal, is to have biochemical and physical characterization (including purity and stability) of the sample supporting the need for neutron studies. A general discussion of small-angle neutron scattering (SANS) is beyond the scope of this article. A brief but thorough introduction is available in the reference work Characterization of Materials by Kaufmann,44 and a comprehensive textbook focused on biological small-angle solution scattering has recently been published.45 Further recommended reading is given in the Discussion section. Small angle scattering uses the so called scattering vector Q as the central quantity that describes the scattering process. This article uses the widely accepted definition Q = 4&#960; sin(&#952;)/&#955;, where &#952; is half the angle between incoming and scattered beam and &#955; is the wavelength of the neutron radiation in Angstroms. Other definitions exist that use different symbols such as &#39;s&#39; for the scattering vector, and that may differ by a factor 2&#960; or by using nanometers in place of Angstrom (see also discussion of Figure 10).

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Amicon Ultra MWCO 50KDa concentrator&#160;</td>
			<td style="width:209px;">EMD Millipore</td>
			<td style="width:108px;">UFC905096</td>
			<td style="width:135px;">labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium citrate dibasic</td>
			<td>Fisher Scientific</td>
			<td>A663</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium sulfate</td>
			<td>EMD Millipore</td>
			<td>2150</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bioflo 310 Bioreactor System</td>
			<td>Eppendorf</td>
			<td>M1287-2110</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>Acros</td>
			<td>423525000</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>IBI Scientific</td>
			<td>IB02025</td>
			<td>antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloramphenicol</td>
			<td>EMD Millipore</td>
			<td>3130</td>
			<td>antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cobalt (II) chloride</td>
			<td>Acros</td>
			<td>AC21413-0050</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Copper (II) sulfate</td>
			<td>Acros</td>
			<td>AC19771-1000&#160;</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>756822</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Drierite Gas Purifier</td>
			<td>W.A. Hammond Drierite Co. Ltd.</td>
			<td>27068</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA, disodium, dihydrate</td>
			<td>EMD Millipore</td>
			<td>4010</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Emulsiflex-C3</td>
			<td>Avestin</td>
			<td>EF-C3</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#196;kta Purifier UPC100</td>
			<td>GE Healthcare&#160;</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiPrep 16/60 Sephacryl S-300 HR column</td>
			<td>GE Healthcare&#160;</td>
			<td>17116701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>VWR</td>
			<td>97064-622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>Teknova</td>
			<td>I3325</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iron(III) chloride hexahydrate</td>
			<td>MP Biochemicals</td>
			<td>ICN19404590</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Agar Miller</td>
			<td>Fisher Scientific</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate heptahydrate</td>
			<td>VWR</td>
			<td>97062-134</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Manganese(II) sulfate monohydrate</td>
			<td>Acros</td>
			<td>AC20590-5000</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MaxQ 6000 Incubated/Refrigerated Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7&#160;</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">n-Dodecyl-d25-&#946;-D-maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310T</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">n-Dodecyl-&#946;-D-maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium phosphate monobasic</td>
			<td>VWR</td>
			<td>97062-346</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific Sorvall</td>
			<td>46910</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SIGMAFAST protease inhibitor cocktail tablets, EDTA-free</td>
			<td>Sigma-Aldrich</td>
			<td>S8830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>795429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 25mm syringe filter with 0.2&#181;m PES membrane</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td>labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable bottle top filter with 0.2&#181;m PES membrane</td>
			<td>Thermo Scientific</td>
			<td>596-4520&#160;</td>
			<td>labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superdex 200 10/300 GL&#160;</td>
			<td>GE Healthcare&#160;</td>
			<td>17517501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superose-12 10/300 GL column&#160;</td>
			<td>GE Healthcare&#160;</td>
			<td>17517301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrospec 10 Cell Density Meter</td>
			<td>GE Healthcare&#160;</td>
			<td>80211630</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate monohydrate</td>
			<td>Acros</td>
			<td>AC38980-2500&#160;</td>
			<td>medium component</td>
		</tr>
	</tbody>
</table>

contrast-matching detergent in small-angle neutron scattering experiments for membrane protein structural analysis and ab initio modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

A beam time and instrument proposal should clearly convey all information needed to the review committee so that a valid assessment of the proposed experiment can be made. Communication with an NSS is highly suggested for inexperienced users. The NSS can assess initial feasibility and guide proposal submission to emphasize feasibility, safety, and the potential for high-impact science. The information provided in the proposal should include background information and context for the signi...

Watch this Scientific Journal Video about Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling at JoVE.com

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

This method can help answer key questions about integral membrane proteins such as the oligomeric state, their size, overall shape and low resolution structure in solution. Solution studies of membrane proteins requires stabilizing molecules like detergents. This technique has the advantage that it makes detergents invisible and highlights the signal from the membrane protein.The implications of this technique extend to many biological and medical research questions because over 1/3 of proteins reside in membranes where they perform many vital cellular functions. Visual demonstration of this method is critical as the deuterium labeling and neutron scattering steps are difficult to learn because very few institutions offer training in these techniques. To begin, determine neutron scattering length densities or SLDs using the web application MULCh, modules for the analysis of contrast variation data.Open the contrast module by clicking contrast located in the left navigation pane. Provide text for a project title and enter the details below for two components as subunit one and subunit two. Enter the molecular formula for each component in the formula text box.Then enter the volume in cubic angstroms for each component in the box below volume. Finally, enter the details for buffer components. Click submit to perform the neutron contrast calculations.A results page is generated that provides a table of scattering length density and contrast match points as well as formulae for determining these parameters at any given percentage of D2O in the buffer. Review the scattering parameters with particular attention to the component contrast match points. Grow E.coli cells harboring an inducible expression vector for the target protein sequence in LB medium to an optical density at 600 nanometers or OD 600 of approximately one.Adapt E.coli cells to deuterium labeled medium prior to scale up for over expression of a deuterated protein by diluting the LB culture one to 20 in three milliliters of minimal medium. After growing to an OD 600 of approximately one, repeat the one to 20 dilutions using minimal medium containing 50, 75, and 100%D2O. Once adapted, continue growing the culture in a bioreactor to increase the yield of deuterated cell mass.As the D2O content is increased, growth rates will decrease. Proceed to harvest, lyse and purify cells as described in the text protocol. Next equilibrate a size exclusion column with greater than two column volumes of final exchange buffer using a fast protein liquid chromatography system.After injecting the sample, run the column in isolate fractions corresponding to the protein of interest. Reserve an aliquot of matched buffer for small angle neutron scattering or SANS background subtraction. To collect SANS data, first load samples and buffers into quartz cells.After ensuring that the beam shutter is closed, approach the sample environment area and place the quartz cells in the sample changer. Record the sample changer position for each sample cell. Check the area and ensure the beam path is free from obstruction before leaving the sample environment area.Then open the beam shutter. Execute a table scan for automated data collection. Follow the instructions for instrument operation provided by the neutron scattering scientist.Use Mantid Plot software and Python script for the reduction of SANS data from a 2D image to a 1D plot. To do so, log in to the remote analysis cluster and execute Mantid plot from the command line. Then open the provided user reduction script and place the corresponding scan numbers or identification for the sample in buffer pairs in the appropriate list.Execute the script to generate reduced data as a four column text file in the specified location. Right click the appropriate work space and Mantid plot and select plot with errors for an initial examination of the scattering profiles. Download the ATSAS software suite and use PRIMUS for data plotting, buffer scaling and subtraction and Guinier analysis.Launch the ATSAS, SAS data analysis application and load the reduced data files corresponding to the sample and buffer pair. To scale the buffer properly, select the data range at high Q where both profiles are similar in flat and click the scale button located under the operations tab. Increase the data range to view all points, click subtract to perform this operation.Perform a Guinier analysis of the buffer subtracted sample data using the analysis tab of the SAS data analysis application. Be sure the proper file is selected in the list and click radius of gyration. An automated attempt to perform a Guinier fit will be provided by clicking on the auto RG button.Expand the range of data used to include all of the low Q data and begin narrowing the data range by taking away high Q points until the Q max times RG limit is below 1.3. Use the plot of residuals to verify that data are linear in the fit range. Make small adjustments to the fit region and monitor the sensitivity of these values to the range of data used in the fit.Obtain the probability distribution function P of R in gnome. Now start the distance distribution wizard within the analysis tab. Obtaining a good fit to the data is essential to obtaining a quality model.Determine Dmax, the maximum interatomic distance within the molecule. Estimate a value for Dmax by unchecking the box to force Rmax equals zero and entering a large value for Dmax. The first X intercept in the plot of P of R yields this estimate.Make incremental changes to this Dmax value as well as the range of data used for number of points used to optimize the gnome fit to the data and the resulting P of R curve. Start the PRIMUS shape wizard from the dammif button on the analysis tab of SAS data analysis and use a manual selection of parameters. Define the Guinier range from the fit and proceed to the next step using the navigation buttons.Define fit values from the P of R plot and proceed to the next step. Provide parameters for the ab initio modeling process. Then initiate the process using the commit button.Overlay and superimpose the final sans envelope with a related high resolution model using supcomb within ATSAS. Place a copy of the high resolution PDB model to be fit to the SANS envelope in the working directory. Then execute supcomb from the command line using the PDB file names as two arguments with the template structure listed first.Finally visualize the ab initio model results using PyMOL, a 3D molecular graphics viewing program. Once the SANS envelope and high resolution structure have been superimposed, these models can be visualized using any molecular graphics viewing program. An overview of the PyMOL visualization process is shown.Once the PDB structure files have been opened in PyMOL, the models should be visible in the PyMOL viewer window. The representations of each model can be changed using the S button next to each file name. Here a surface representation is used for the SANS envelope and a cartoon representation is used for the protein backbone of the high resolution model.A suitable color scheme can be selected from the options available under the C button. For protein chains, a chainbow coloring provides a color gradient from N to C terminus to aid interpretation. Transparency is applied to the surface representation to allow better visualization of the protein structure within the envelope.For publication images, a white background is recommended. Once these visualization cues have been applied the 3D structures can be examined by clicking and dragging the structure. Manipulations of the perspective and molecule rotation and translation can be performed.While attempting this procedure it's important to remember to accurately match the neutron contrast of the detergent head and tail groups with the D2O H2O solvent. After its development, this technique paved the way for exploring the structure of intramembrane asparto proteases. These proteins are involved in the immune response, hepatitis C and Alzheimers disease.Don't forget that working in biochemical laboratories and at neutron beam facilities can be hazardous. Precautions such as wearing personal protective equipment and following all facility safety rules and radiological postings are mandatory while performing this procedure.

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