eamag/protocols-with-steps huggingface dataset:
TITLE: Fluorescence size exclusion chromatography (FSEC) from ATP13A2 microsomes
AUTHORS: Sue  Sim, eunyong_park  

[DESCRIPTION]
Using fluorescence size exclusion chromatography (FSEC) to analyze ATP13A2 expression in microsomes

[STEPS]
1. Thaw 100 μg microsomes on ice
2. Resuspend microsomes in Lysis Buffer and final volume 1% DDM/0.2% CHS (1X pellet, 3X Lysis Buffer, 1X 5% DDM/1% CHS) at 4 °C 

2.1. DDM: n-dodecyl-β-D-maltopyranoside (Anatrace)
2.2. CHS: cholesteryl hemisuccinate (Anatrace)
3. Solubilize by rotating end-over-end for 2 h at 4C
4. Clarify lysate by spinning at 17000 x g, 60 min, 4 °C 

5. Equilibrate Superose 6 column, connected to an HPLC system, with Running Buffer
6. Inject 85 uL of lysate into HPLC
7. Monitor elution of GFP-tagged ATP13A2 constructs using a fluorometer (λex= 475 nm; λem= 510 nm; with a fixed gain) connected to the system
TITLE: Phosphate Buffered Saline (PBS)
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Phosphate Buffered Saline (PBS)]
Prepare  by dissolving  , and  in .
150 mM NaCl10 mM Potassium Phosphate buffer
[PBS]
[NaCl]
[K2HPO4 • 3H2O]
[KH2PO4]
[distilled water]
?. [Phosphate Buffered Saline (PBS)]
Adjust the pH before use.
?. [Phosphate Buffered Saline (PBS)]
A variation of PBS can also be prepared as follows:
[NaCl]
[KCl]
[Na2HPO4]
[KH2PO4]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[GUIDELINES]
<p><em>Note: For Human CD4 Nanobeads, please refer to the protocol found on the product webpage for MojoSort™ Human CD4 Nanobeads (Cat. No. 480013/480014).</em></p>
<p><strong> </strong></p>
<p><strong>Reagents and instruments required</strong>:</p>
<p><br />-MojoSort™ Buffer (5X) (Cat. No. 480017)<br />-MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />-Adjustable pipettes<br />-70 μm filters (one per sample)<br />-5 mL (12 x 75 mm) polystyrene tubes<br />-Reagents for sample preparation<br />-Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><strong>Protocol:</strong></p>
<p>This protocol has been optimized to remove washing steps after nanobeads incubation, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dnyecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dnxecje.jpg" data-ofn="MojoSort_nowash.JPG" /></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Starlet Sea Anemone (Nematostella vectensis) 2021 Environmental Summary, Reptile &amp; Aquatics, Stowers Institute for Medical Research
AUTHORS: Shane  C. Miller, Diana P Baumann, M.  Shane Merryman

[DESCRIPTION]
The starlet sea anemone (Nematostella vectensis) is an emerging model organism, and we have maintained a colony at the Stowers Institute since 2007. Nematostella are known as a simple sea anemone, related to other cnidarians such as jellyfish and corals. Native to estuarine environments across the Atlantic coast of North America, from Novia Scotia to Florida, they encounter a variety of environmental conditions (e.g., temperature, salinity). Acknowledging that husbandry conditions and environmental parameters can impact research results we provide information about the housing, nutrition, maintenance, and health for our colony of Nematostella. This information will be applicable to any Nematostella housed in the facility in 2021.

[STEPS]
TITLE: ADP-Glo kinase assay
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">An assay for measuring ULK1 kinase activity</div></div>

[STEPS]
?. Make a mixture of 100 nM purified wild-type or kinase dead ULK1 complex with 5 µM ULKtide in the presence or absence of 100 µM ATP. The total volume is 50 µL.
?. Add 50 µL ATP-depletion reagent to terminate the reaction and deplete the unconsumed ATP.
?. Incubate at room temperature for 40 min.
?. Add 10 µL of kinase detection reagent to convert ADP to ATP and introduce luciferase and luciferin to detect ATP.
?. Measure the luminescent output  by a GloMax-Multi detection system.
?. Incubate at room temperature for 1 h.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Yeast is a well-characterized genome due to its small size and historical significance in genetics.  The website http://yeastgenome.org/ is a dedicated resource for yeast genomics.</p>
<p> </p>
<p>For this exercise, I want you to create a "Makefile" that will execute this entire pipeline if I type "make."  </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Protein Transfer using Bio-rad TransBlot Turbo with Turbo RTA Transfer Kit
AUTHORS: Lynn   Doran, Steven J Burgess

[DESCRIPTION]
A protocol for transfer of proteins from acrylamide gels onto nitrocellulose membrane for immunoblot analysis. This protocol is based on the assumption that TGX pre-cast gels have been used, the procedure is very similar for self-made gels, just that the running time is longer. Using the transfer packs is economically similar to wet-transfer using MeOH.  

Note:  Clean the apparatus immediately after use with dH2O and dry. This prevents the build-up of salts and rust which impair performance.  

Literature: 
Full manual: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10020688.pdf 
Quick-start: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10016505D.pdf 
Transfer pack: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10019593D.pdf

[STEPS]
6. Place the  gel  in the middle of the membrane and roll to remove air-bubbles.

 

7. Place the top stack on top of the gel, gently roll

8. Close the cassette lid, taking care not to disturb the gel
9. Place the cassette in the Trans-Blot Turbo and follow the instructions on the machine (Fast protocol TGX gel - 3 minutes). 
 

10. Either dry membrane and store at 4 °C for later use or proceed immediately to fluorescent western protocol

1. Wet and equilibrate the membrane.
    Nitrocellulose: 2-3 min in 1X transfer buffer.
    PVDF: Soak in methanol until translucent than transfer to 1X transfer buffer for 2-3 min.
2. Soak transfer stacks of pads (separated by blue paper) side by side in 1X transfer buffer for 2-3 min.
3. Following SDS-PAGE electrophoresis, remove the pre-cast gel by using the small green Bio-rad lever tool to crack apart the cast at each arrow.  Gently remove one side of the plastic cast.  Use the green Bio-rad scraper to cut off the "arms" that formed the wells and the bottom edge of the gel that was below the junction of the two cast pieces.
4. Using tweezers, move a stack of transfer pads to the cassette.  Use the wetted, small roller to smooth the transfer pads.
5. Using tweezers, move the membrane to the center of the transfer pads in the cassette.  Use the wetted, small roller to smooth the membrane.
TITLE: LRRK1 expression and purification
AUTHORS: Yu Xuan  Lin, janice reimer

[DESCRIPTION]
Protein purification protocol for full-length LRRK1 as done by Leschziner and Reck-Peterson Labs.

Original protocol by Janice M. Reimer and Yu Xuan Lin.

[STEPS]
SECTION: His6-Z-TEV-LRRK1 Purification
1. N-terminally tagged His6-Z-TEV-LRRK1(FL) was expressed in Sf9 insect cells. Insect cells were infected with baculovirus and grown at 27°C for 3 days.
SECTION: His6-Z-TEV-LRRK1 Purification
2. Cells were harvested and then resuspended in lysis buffer (50mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP, 0.5 mM Pefabloc and cOmplete EDTA-free protease inhibitor cocktail (Roche).

SECTION: His6-Z-TEV-LRRK1 Purification
3. Cells were lysed using a Dounce homogenizer and clarified by centrifugation at 50000 rpm, 4°C, 01:28:00, and Ti70 rotor.
SECTION: His6-Z-TEV-LRRK1 Purification
4. The supernatant was incubated for 1 hr with Ni-NTA agarose beads (Qiagen) equilibrated in lysis buffer. Beads were applied to a gravity column where they were extensively washed with lysis buffer,  wash buffer ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP), followed by elution in lysis buffer containing 300 mM imidazole ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP, 300mM imidazole)
SECTION: His6-Z-TEV-LRRK1 Purification
5. The eluted protein was diluted to 250 mM NaCl using dilution buffer (50mM HEPES pH 7.4, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP) and loaded onto a SP Sepharose column (Cytiva) equilibrated in buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP) 
SECTION: His6-Z-TEV-LRRK1 Purification
6. The protein was eluted using a 250 mM to 2.5 M NaCl gradient. Fractions containing LRRK1(FL) were pooled, diluted to ~500 mM NaCl, and incubated with TEV protease overnight at 4°C.
SECTION: His6-Z-TEV-LRRK1 Purification
7. The protein was concentrated and put directly over a S200 size exclusion column (Cytiva) equilibrated in storage buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2 and 20 μM GDP).
SECTION: His6-Z-TEV-LRRK1 Purification
8. The protein was concentrated to ~5-6 μM and flash frozen in liquid nitrogen for storage. N-terminally tagged His6-Z-TEV-LRRK120-2015 was purified using the same protocol.
TITLE: 96-well Plate Growth Curve Setup
AUTHORS: Norman van Rhijn, Panagiotis Papastamoulis, Takanori Furukawa, Magnus Rattray, Elaine Bignell, Mike Bromley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Generally, growth assays for filamentous fungi have been performed on solid media, either as dilution series or spot tests. However, the solid media environment does not accurately mimic the environment encountered during infection (ie the mammalian lung). Previously, we have developed a methodology to perform liquid growth assays in time for </span><span style = "font-style:italic;">A. fumigatus </span><span>and other filamentous fungi including analysis via mathematical modelling.</span></div><div class = "text-block"><span>This protocol is designed for </span><span style = "font-style:italic;">Aspergillus </span><span>species (and other filamentous fungi) to generate growth curves in liquid media in a 96-well plate. This can be done in high-throughput to generate 96 growth curves per run.</span></div></div>

[STEPS]
?. [Plate preparation]
Harvest and dilute spores in PBS+0.01% Tween to 4*105 spores/mL.
?. [Plate preparation]
Aliquot 5 uL of this spore stock per well of a CytoOne 96-well plate (Starlab) non-coated, this will be overlayed with 195 uL of liquid media to a total volume of 200 uL.
This equals to 2000 spores (absolute) per well. 1000 spores or 500 spores are possible to use, but result in more variance within replicates.
?. [Machine setup]
In the Manchester Fungal Infection Group we either use a Powerwave X-2 with KC software or a BioTek platereader. Set this up to run for   at   to measure OD600 every 10 minutes. Take a blank reading before running the plate and preheat the machine. Run your experiment.This will result in a "growth curve".Extract or manipulate the data to look like attached (txt file format).
37 °C
?. [Plate preparation]
Fill the space between the wells with either media or H2O. This will avoid an "edge effect", where the wells around the edge of the plate show increased growth compared to other wells.
?. [Plate preparation]
Cover the 96 well plate with a breathable cover to allow gas exchange.
?. [Analysis]
See our next protocol for analysis.
TITLE: A protocol for computerized quantitative analysis of nerve fibers, mast cells, enteric glial cells and the proximity of mast cells to the nerve fibers in 3D Images of human sigmoid mucosal biopsies
AUTHORS: Tao Li, Pu-Qing Yuan, Yvette Taché

[DESCRIPTION]
This protocol describes a step-by-step computational workflow that we developed by adapting
Imaris 9.7-9.9 Surfaces Rendering Technology (https://imaris.oxinst.com/products/imaris-for-neuroscientists) to perform the quantitative analysis of nerve fibers, enteric glial and immune cells in 3D images of human sigmoid mucosal biopsies, as well as to assess the proximity of mast cells to the nerve fibers which cannot be easily portrayed and precisely measured with 2D images. The volumes of surface-masked nerve fibers and enteric glial cells, the numbers of spotted mast cells and the shortest distances of the centers of each individual spot to the surfaces of nerve fibers in 3Dimages were automatically computed and plotted correspondently with Imaris 9.7-9.9. This computerized protocol not only reduces the biases due to observer/examiner judgment and overcomes limitations of 2D images, but also is much faster than measuring
manually and allows us to quantitate a larger number of samples, increasing statistical accuracy. The parameters created in this protocol also provide efficient reference for us to apply the New AI Machine Learning Segmentation implemented into Imaris 10.1 recently for our quantitative analysis of large datasets.
The study using this protocol is relevant to the underlying peripheral mechanisms of visceral pain in irritable bowel syndrome.

[STEPS]
1. 
2.
TITLE: A Comparison of the Performance of Disinfection Agents on Smallmouth Grunt (Haemulon chrysargyreum) Eggs
AUTHORS: Tory Stoddard, Quint Heyder, Alyssa Leonardi, Andrew Saltzman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">    Fish egg disinfection is a common practice in aquaculture facilities to prevent disease and improve the hatch rates of fish eggs. This process helps remove bacteria, fungus, and other unwanted organisms from eggs. Iodine was found to be the most effective disinfecting agent based on available literature and recent experimentation. </div></div>

[STEPS]
?. Separate eggs from aerated collection bucket using a 40μm siv.
?. Pipette a few milliliters of eggs from the 40μm siv into a 10mL graduated cylinder.
?. Prepare the disinfectant bath by filling the watch glass halfway with filtered seawater. Then, add 1mL of iodine per 1L of filtered seawater.
?. Once the eggs have settled and floated to the top, measure the eggs volumetrically and pipette 0.3mL of eggs out.
?. Prepare another watch glass filled with just filtered seawater.
?. After 5 minutes rinse the eggs in filtered seawater by placing the siv from the treatment bath into the watch glass filled with filtered seawater only. Similar to the treatment bath, carefully and slowly swirl the siv in a circular motion in the filtered seawater bath for about 30 seconds.
?. Place a 40μm siv into the iodine treatment bath and pipette the 0.3mL of eggs into the siv. Carefully and slowly swirl the siv in a circular motion in the treatment bath for about 30 seconds to mix the contents. Allow the eggs to be exposed to the iodine treatment for 5 minutes using a timer.
?. Pipette ~0.1mL of eggs from the siv into each of the three watch glasses containing filtered seawater.
?. Place the three watch glasses of eggs into the heated trough.
?. Make sure to pour the leftover eggs back into the collection bucket, so they do not get deprived of oxygen.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>The MELD Project is an international collaboration aiming to create open-access, robust and generalisable tools for FCD detection. To this end, we will train a neural network classifier on MRI features from FCD patients from multiple centres worldwide.</em></p>
<p><strong> </strong></p>
<p><strong>Protocol 5 provides instructions </strong><strong>for post-processing FreeSurfer reconstructions to create final feature matrices.</strong></p>

[BEFORE_START]
<p>These steps require FreeSurfer and Python</p>
<p> </p>
<p><strong><u>FreeSurfer</u></strong></p>
<p>Instructions for installing Freesurfer are included in MELD Protocol 2</p>
<p> </p>
<p><strong>NB:</strong><strong> FreeSurfer and its utilities need to be in your path or this script will not run properly. You can type tksurfer on the command line to make sure it is available.</strong></p>
<p> </p>
<p>Before you want to work with FreeSurfer, you must make sure three things have happened:</p>
<ol>
<li>The variableFREESURFER_HOME is set (so your computer knows where FreeSurfer is installed):</li>
</ol>
<p> </p>
<table>
<tbody>
<tr>
<td width="423">
<p><em>setenv FREESURFER_HOME &lt;</em><em>freesurfer</em><em>_installation_directory&gt;/freesurfer</em></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<ol start="2">
<li>The FreeSurfer set up script must be sourced (so FreeSurfer knows the location of everything it needs):</li>
</ol>
<p> </p>
<table>
<tbody>
<tr>
<td width="284">
<p><em>source $</em><em>FREESURFER</em><em>_HOME/SetUpFreeSurfer.csh</em></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<ol start="3">
<li>FreeSurfer has been pointed to a directory of subjects to work on:</li>
</ol>
<p><em> </em></p>
<table>
<tbody>
<tr>
<td width="229">
<p><em>setenv SUBJECTS_DIR &lt;path&gt;/meld/output</em></p>
</td>
</tr>
</tbody>
</table>
<p> </p>

[GUIDELINES]
<p><strong>If you have any questions or run into problems, please feel free to contact the MELD project: (meld.study@gmail.com)</strong></p>

[STEPS]
?. 
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?. 
?. 
?. 
?.
TITLE: Preparing whole cell samples for immunoblot analysis
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for preparation of HeLa cell lysates for immunoblot analysis.

[STEPS]
1. Add an appropriate volume of 1x LDS Sample Buffer (ThermoFisher) to each sample.
2. Boil each sample at 99 °C with shaking at maximum speed for 10 min 

3. Allow all samples to cool to Room temperature, and quickly centrifuge the samples to collect all liquid in the bottom of the tube. Vortex each sample for ~3 seconds to ensure homogeneity. 

4. Measure the concentration of each sample spectroscopically, blanking with the 1x LDS sample buffer and using an A280 measurement. Dilute samples with 1x LDS Sample buffer to a concentration of < 7 mg/mL if required. 
5. Aliquot out the desired amount of each sample into a separate tube, and add 1x LDS Sample Buffer to each sample to make all samples in the same gel a standard volume. 
6. Either freeze samples at -20 °C until required, or load directly onto a SDS-PAGE gel.
TITLE: Chlamydia trachomatis detection and gene expression protocols 
AUTHORS: Athumani M. Ramadhani, Tamsyn Derrick, David Macleod, Patrick Massae, Aiweda Malisa, Kelvin Mbuya, Tara Mtuy, William Makupa, Chrissy h Roberts, Robin L. Bailey, David C. W. Mabey, Martin J. Holland, Matthew J. Burton

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?.
TITLE: PMN- 05 Culture of Human PMN - IL-8 production
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Published work using this protocol:</div><div class = "text-block">- Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes.</div><div class = "text-block">Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.</div></div>

[STEPS]
?. Isolate PMN according either to the protocol "PMN- 01a" or "PMN- 01b".
?. Resuspend PMN at 10x106 cells/ml in complete culture medium (SOLUTION- 13).
?. Use a number of tubes according to the experimental plan and to the substances to test.
?. Tubes will be prepared to test resting IL-8 production (substances alone) and stimulus-induced production.
Add substances before the activation with stimulus (time selected according to the experiental plan)
Stimulus: fMLP 0.1 μM or LPS 1 μM or other.
?. Incubate PMN atfor in a 5 ml tubes for cell culture. Cells will be incubated in resting conditions or in presence of stimulus (point 3-4).
37 °C
?. Centrifuge at
Centrifuge: 1500 34, 10 min
?. Harvest cell-free supernatant using a pipette and cell pellet (for real-time assay of mRNA for IL-8).
?. Store the supernatant and cell pellet at -80°C until the IL-8 Enzyme Linked-ImmunoSorbent Assay (ELISA) and/or real time assay is performed.
TITLE: Glass bead transformation of Heterosigma akashiwo
AUTHORS: Deepak Nanjappa and Kathryn Coyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was developed for the glass bead-mediated transformation of </span><span style = "font-style:italic;">Heterosigma akashiwo</span><span>. This species does not grow on solid medium, so transformants are grown "in bulk" and should not be considered clonal. We have repeated this protocol with success and have been able to maintain transformants on selection medium for several months. </span></div></div>

[STEPS]
?. Harvest exponential growth Heterosigma akashiwo culture (1-2 x 106 cells mL-1) by centrifugation at 1500 rpm for 90 s in a 50 mL sterile screw-cap tube. Remove the supernatant by pipetting.
?. Resuspend the cell pellet in a small volume of MAX Efficiency® Transformation Reagent for Algae (Invitrogen, Thermo Fischer Scientific, USA) or in 384mM Sorbitol.
?. Transfer 300µl of cells to a 1.5 ml microcentrifuge tube and add up to 50 µl of linearized plasmid DNA (1 – 10µg). Mix by tapping the mixture and incubate for 10 minutes at room temperature. Plasmid sequences can be found at http://doi.org/10.5281/zenodo.439653.
?. Weigh approximately 300 mg of glass beads (425-600µm, Sigma-Aldrich, USA) into a 2 ml microcentrifuge tube and add the cells-DNA mixture to the glass beads. Vortex at full speed for 10 secs and let it sit on bench for 15 mins.
?. Transfer the mixture in equal amounts to 4 wells of 12 well plate with 2 ml of fresh f/2 medium with cefotaxime (100µg/ml, to control bacterial growth).
?. Incubate in dim light for 1 day and then transfer to normal light conditions for another 2 days. After 3-4 days, add the appropriate antibiotic and incubate under normal conditions.
?. [Expected Results]
Transformants should be visible after 20-30 days. Continue to transfer the live cells to fresh medium every 10-12 days with antibiotic and analyse transformants by PCR once sufficient culture is obtained.