eamag/protocols-with-steps huggingface dataset:
TITLE: Preparing mRNA for nucleofection of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure for preparing mRNA to be delivered into human pluripotent stem cells (hPSCs) using nucleofection.

General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.



[STEPS]
1. Thaw mRNA and synthetic pegRNA/ngRNA on ice 

2. In each nucleofection, use 4 µg total of mRNA
3. For prime editing PE2 strategy, use:
    4 µg, IVT PE2 mRNA
    1.5 µl, 100 µM Synthetic pegRNA
4. For prime editing PE3 strategy, use:
    4 µg, IVT PE2 mRNA
    1 µl, 100 µM Synthetic pegRNA
    0.5 µl, 100 µM, Synthetic ngRNA
5. Pipet the proper amount of each component into a microcentrifuge tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Ultracentrifugation provides a means to concentrate, analyze, and purify viruses in solution, and therefore represents an invaluable tool for aquatic virologists. This protocol provides a method for purification of viruses from culture lysates from natural water samples.

[GUIDELINES]
<strong>Materials and reagents</strong><br /><br /> • OptiPrep (60% iodixanol solution)—Axis-Shield, Accurate Chemical and Scientific (Westbury), Progen Biotechnik, or Sigma Aldrich <br />• Open-topped ultracentrifuge tubes—i.e., Beckman Coulter Ultra-Clear <br />• Ultracentrifuge. <br />• Swing-out Ultracentrifuge Rotor—i.e., Beckman Coulter SW41, SW28, or MLS50 <br />• 30 kDa cutoff disposable centrifugal ultrafiltration devices—i.e., Millipore <br />• 3-mL syringe with Luer-Lok or Luer-Slip <br />• Pipetting needle—i.e., Cadence Science, stainless-steel 14- or 16-guage 4-inch cannula with Luer hub or Slip hub <br />• Sterile 1.5 mL microcentrifuge tubes for collecting gradient fractions <br />• Sterile disposable transfer pipettes <br />• Sterile virus-free media for resuspending and diluting virus <br />• Polyethylene glycol, average molecular weight 6000- 8000—i.e., Fisher Scientific Carbowax PEG 8000, or Sigma Aldrich Biochemika Ultra 8000<br /><br /><strong>Discussion</strong><br /><br />OptiPrep must be removed from samples before examination of virus particles by negative staining and TEM. This can be achieved using disposable Millipore centrifugal ultrafiltration devices with a 30 kDa cutoff. For most other applications OptiPrep does not need to be removed prior to further analysis, although it should be assayed to determine effects on the growth of specific viral-hosts when re-infection assays are used to confirm purification of the infectious agent.

[STEPS]
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TITLE: His-tag purification 
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">His tag purification uses the technique of immobilised metal affinity chromatography. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. It has been studied that among amino acids constituting proteins, histidine is strongly involved in the coordinate bond with metal ions. Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out. When a protein having a His tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier, they can be washed off with a buffer.  Thereafter, it is possible to recover the protein having the His tag with high purity. </div></div>

[STEPS]
?. [Separate proteins from soil matrix]
Collect soil samples of
5 g
?. [Separate proteins from soil matrix]
Extract total proteins using NoviPure Soil Protein Kit or other  comercially available kits for total protein soil extraction
?. [His-tag separation of NLP]
Wash the Ni2+-sepharose column material with 12 CVs of MQ and 4 CVs of Column Wash Buffer (imidazole, KPi , NaCl ).
Use ±0.5 ml of Ni2+-sepharose column material per 10 mg of total protein.
?. [His-tag separation of NLP]
Apply the sample, add imidazole (10mM final concentration) and the washed Ni2+-sepharose column material. Nutate in at  for
4 °C
?. [His-tag separation of NLP]
Pour column, collect flow through to apply on SDS gel.
?. [His-tag separation of NLP]
Wash column with 20 CVs of Wash Buffer ( imidazole, KPi , NaCl ).
?. [His-tag separation of NLP]
Elute protein with Elution Buffer ( imidazole, Kpi , NaCl ) in  fractions. Check elution fractions Absorbance by NanoDrop.
200 µl
?. [His-tag separation of NLP]
Run an SDS gel to check purification:- Soil suspension & Flow through: dilute 15x, apply- Wash: dilute 1.25x, apply - Elution fractions: dilute to ±0.2 mg/ml, apply
5 µl
10 µl
5 µl
TITLE: Automatic flow in fluid-walled dumbbells driven by Laplace pressure
AUTHORS: Quyen Do, Federico Nebuloni, Richard Wade-Martins

[DESCRIPTION]
This protocol describes experiments performed to quantify pressure and volume variations inside fluid-walled dumbbells when a pressure difference is generated between the two chambers. Difference in Laplace pressure automatically flows medium from the high-pressure chamber to the low pressure one. Flow stops when pressures are equilibrated.

[STEPS]
SECTION: Jet-Printing of Fluid-Walled Dumbbells
1. Fill a virgin uniwell plate with 5 ml of DMEM supplemented with 10% FBS. Agitate the plate to spread the 5 mL of medium to cover the whole area of the plate.
SECTION: Jet-Printing of Fluid-Walled Dumbbells
2. Remove the volume leaving a thin layer of medium wetting the plate. 
SECTION: Jet-Printing of Fluid-Walled Dumbbells
3. Gently overlay FC40 (~50 mL) pouring it in one of the corners. 

SECTION: Jet-Printing of Fluid-Walled Dumbbells
4. Place the plate in the 3D traverse. Adjust x, y, z positions of the traverse head to coincide with the starting point of the movement path. 
SECTION: Jet-Printing of Fluid-Walled Dumbbells
5. Start the pump, setting a constant flow at 480 μL/min.
SECTION: Jet-Printing of Fluid-Walled Dumbbells
7. Start traverse automatic movement. 
 

SECTION: Jet-Printing of Fluid-Walled Dumbbells
8. At completion of the movement path, stop pump and remove plate from the traverse. 
 

SECTION: Establishment of Pressure Difference and Volumes Variation Measurements
9. Place the newly printed plate into the pendant drop tensiometry machine.
SECTION: Establishment of Pressure Difference and Volumes Variation Measurements
10. Add FC40 to almost fill the plate entirely (~20 mL).
SECTION: Establishment of Pressure Difference and Volumes Variation Measurements
11. By mean of a syringe pump connected to a needle through a Teflon tube, infuse 4 μL in the right chamber and soon after 1 μL in the left chamber (infusion rate set on pump = 20 μl/min).
SECTION: Establishment of Pressure Difference and Volumes Variation Measurements
12. Record chambers height for 24 hours imaging every 30 minutes.
SECTION: Jet-Printing of Fluid-Walled Dumbbells
6. Wait ~10 s to allow flow to be fully developed.
TITLE: PROTAC Design
AUTHORS: boc.protac  

[DESCRIPTION]
PROTAC (protein degradation targeted chimera) is a special protein degradation technology, which uses ubiquitin proteasome pathway, a natural protein degradation pathway in cells, to remove specific proteins that need to be degraded. A PROTAC molecule consists of two key domains: the domain that specifically binds to the target protein that needs to be degraded, and the domain that binds to the E3 ubiquitination ligase, which is linked by a specific linker. Unlike traditional small molecule inhibitors, which can only regulate the activity of target proteins, PROTAC can directly degrade target proteins through the protein destruction mechanism of cells themselves. Intracellular proteins with abnormal structure or function are labeled with polyubiquitin by a series of enzymes, which are then transferred to the proteasome for degradation.

As a leading service provider in drug discovery and research, BOC Sciences is fully capable and committed to providing one-stop proteolysis targeting molecular drug discovery based on chimeric (PROTAC®). This has become a promising strategy in the field of drug discovery. With a comprehensive and advanced platform, we provide PROTAC molecular discovery package services to customers around the world to meet new drug discovery goals. This includes a variety of services that cover the entire process.

[STEPS]
TITLE: Insulin-secreting human alpha-cells might be a cure for diabetes
AUTHORS: Cathy Miller

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">From one hormone to another, it’s usually a long-term change. To explore whether human cells have this ability to adapt, the Herrera team uses islets from diabetic donors and non-diabetic donors. They first classified different cell types, focusing on two of them: alpha cells (cells that produce glucagon) and sputum cells (cells that produce pancreatic polypeptide). Herrera explains, “We divided the cells we obtained into two groups: in one group of cells, we introduced only one fluorescent cell tracer, and in another group, we added a gene of β-cell-specific insulin transcription factor." </div><div class = "text-block"><span>Nowadays, Peptide-based treatment is becoming increasingly important for diabetes treatment, including </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ghrelin Peptides</span></a><span>and Glucagon Like Peptides.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for PFA fixation of sediment or culture samples and Percoll prep of sediments. </p>

[STEPS]
SECTION: 4% PFA in PBS
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: 4% PFA in PBS
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment or culture samples (lab based)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Fixing sediment samples (shipboard)
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
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SECTION: Mini Percoll prep for microscopy
?. 
SECTION: Mini Percoll prep for microscopy
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SECTION: Mini Percoll prep for microscopy
?.
TITLE: Fura-2 imaging of ionomycin response, with and without R568; a CaSR positive modulator
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Fura-2 imaging protocol to accompany Kilfeather, Khoo et al., 2023: Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease



[STEPS]
SECTION: Data Capture
1. Prepare Fura-2 AM to 5 mM concentration in calcium-free HBSS supplemented with 20 mM HEPES.
SECTION: Data Capture
2. Dilute Fura-2 AM to the final concentration of 2.5 mM in Neurobasal medium supplemented with B27 and L-glutamine, with DMSO (0.1%) or the CaSR positive modulator R568 (10 mM).
SECTION: Data Capture
3. Incubate iPSC-derived dopaminergic neurons (DAns) in the solution of Fura-2 AM and drug for 1 h at 37°C, 5% CO2 and then image on a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 37°C.
SECTION: Data Capture
4. Excite the dye at 340 nm and 380 nm and detect at 510 nm. Image each well every 4 s for 100 s and inject with ionomycin (final concentration 5 mM) after a baseline of 28 s.
SECTION: Analysis
5. For the analysis, compute the 340/380 ratio, and then subtract the baseline from all the timepoints, to obtain a normalised trace for each well. Find the maximum intensity (peak amplitude) of the normalised trace and calculate the area under the curve (AUC) using the left rectangular approximation method.
TITLE: Immunoprecipitation for Flag-tagged proteins
AUTHORS: Jean-Louis Parmasad

[DESCRIPTION]
Immunoprecipitation for Flag-tagged proteins for various biomolecular analyses.

[STEPS]
SECTION: Transfection and collection
1. Transfect HEK293T cells with 2µg of plasmid expressing Flag-tagged protein of interest using enhanced polyethyleneimine transfection reagent in Opti-MEM (Thermo Fisher Scientific, 31985070).
SECTION: Transfection and collection
2. After 48h, collect cells and lyse with IP Lysis Buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP4O, 5% Glycerol). 
SECTION: IP
4. Use 3% of the collected sample for the ‘Input’.
SECTION: IP
5. Incubate the remaining sample with Anti-FLAG M2 Magnetic beads (Millipore Sigma, M8823) for 1 h at 4 °C in a revolving rack.
SECTION: IP
7. Wash beads with IP lysis buffer.
SECTION: IP
8. Add 2x Laemmli buffer (Bio-Rad, 1610747, Hercules, CA, USA) with 20% 2-mercaptoethanol (Bio-Rad, 1610710) to the beads. 
SECTION: IP
9. Vortex, then incubate for 15 min at 75 °C to elude proteins from the beads.
SECTION: IP
11. Run all samples on SDS-PAGE gel and visualize with method of choice.
SECTION: Prepare beads
3. Defrost beads on ice. Wash 3x with IP Lysis Buffer.
SECTION: IP
10. Place tubes in magnetic rack and collect elution.
SECTION: IP
6. Place tubes in magnetic rack and collect 3% the supernatant as the “Flow-through” sample. Aspirate the remaining flow-through.
TITLE: Adhesive Test
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Adhesive behavioural test.

[STEPS]
1. Habituate mice in the testing room for 60 minutes.
2. Remove all mice from their home cage then remove all bedding/debris from their home cage (do not clean their home cage, just give a light wipe with a dry paper towel).
3. Place one mouse back into its home cage for 1 minute.
4. Scruff mouse and place 1cm2 adhesive on each forepaw then place the mouse back into the home cage. Record its movement for 2 minutes, calling out or noting when it contacts and removes the adhesive from each forepaw.
5. Once each piece of tape is removed or 2-minutes is reached, whichever comes first, remove the mouse and place it back into the holding cage.
6. Repeat for all mice of that cage then replace the bedding and move mice back into their home cage.
TITLE: In vitro LRRK2 kinase activity assay using mass-spectrometry as readout
AUTHORS: Verena Dederer, chatterjeedeep, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine in vitro substrate phosphorylation for any purified kinase substrate pair.

It can be used to determine inhibition curves by addition of concentration series of kinase inhibitors.

Reaction is carried out in 50 µL reaction mix containing final concentration of 50 nM kinase and 5 mM substrate.

[STEPS]
6. Incubate reaction for 3 h at room temperature.
Note: depeding on kinase this may be shorter or longer.
SECTION: Step-by-Step Protocol
1. Prepare 50 µL reaction mix I: 50 nM purified LRRK2 and 5 µM Rab8 substrate in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.
Aliquot 25 µL per tube.


 

SECTION: Step-by-Step Protocol
3. Prepare 25 µL reaction mix III: same as reaction mix II but no ATP as negative control.
SECTION: Step-by-Step Protocol
4. Start reaction by adding 25 µL reaction II to reaction mix I.
Note: for negative control add 25 µL reaction mix III to second aliquot of reaction mix I.
SECTION: Step-by-Step Protocol
5. Mix by vortexing and brief centrifugation for 30 sec with 500xg.
SECTION: Step-by-Step Protocol
2. Prepare 25 µL reaction mix II: 2 mM ATP + 2.5 mM MgCl2 in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.

 

7. Stopp reaction by adding 50 µL mass spec buffer (dH2O+0.1% formic acid).
8. Store at -80°C or proceed directly with mass spectrometry analysis.
TITLE: Processing of Radial K-space DW-MRI Data
AUTHORS: Miguelrj, Hee Kwon Song, Stephen Pickup, Mamtaaryagupta, Rong Zhou

[DESCRIPTION]
The protocol includes reconstruction of diffusion weighted images from radial k-space data and using a 3-parameter fit to derive metrics of ADC and kurtosis index.

[STEPS]
1. Image reconstruction

Radially acquired diffusion-weighted images (DWIs) are reconstructed using the following steps:

Apply zero-order phase correction to each radial spoke using the average phase offset at the center slice of the lowest b-value image
Zerofill k-space by a factor of 2 to double field of view
Multiply signal of each point by its respective area on a Voronoi diagram of the points (including added zerofill points) in k-space
Re-grid each radially defined point to its nearest Cartesian coordinate using its Kaiser-Besel index
Apply Fourier transform to now Cartesian-defined k-space


This process can be easily done using the DWI Processing resources [link].
To use this, place both DWI_shell.ipynb and DWI_processing_functions.py in the same directory (your working directory). The readme.txt file outlines specific file formats, function inputs, and outputs. 

Quickly, once all files are formatted according to readme.txt, the first cell defines all image and reconstruction parameters:
 

Each cell should then be run consecutively.
TITLE: Cylinder Test
AUTHORS: Xiaobo Mao, Ramhari Kumbhar, Hanseok Ko, Valina L. Dawson, Ted Dawson

[DESCRIPTION]
The Cylinder test is used to evaluate locomotor asymmetry and spontaneous movement in rodent models.

[STEPS]
1. Set up small, transparent cylinder in front of video recorder. 
2. Place mouse in cylinder and record spontaneous activity for 10 min. 
3. Clean cylinder in between mice.
4. For all recordings, view in slow motion and count the number of forepaw touches to the cylinder walls, rears, and grooming bouts.
TITLE: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-sala de estudio?
AUTHORS: Pedro Postigo Gimeno

[DESCRIPTION]
Aplicación de reserva de espacios y como reservar un asiento en la Biblioteca/Sala de Estudio de la Universidad Católica de Murcia UCAM

[STEPS]
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
1. Aplicación reserva de espacios

  
Primeros pasos para utilizar la nueva aplicación de reserva de espacios de la universidad: 

Instrucction in English 
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
1.1. Regístrate con tu email UCAM a través de este enlace: 

https://go.ucam.edu/registro-reserva-espacios
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
1.2. Te llegará un correo electrónico, confirma tu registro y descarga la App Take a Spot.
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
1.3. Ya podrás acceder a la aplicaciónTake a Spot a través de la web o de la aplicación móvil:

       

SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2. ¿Cómo reservar un asiento en la biblioteca-sala de estudio?
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.1. Descarga la app de reserva de espacios Take a Spot.

  
APP Store
Google Play
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.2. Inicia sesión con tu correo UCAM y contraseña habitual en la app.
        
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.3. Selecciona el Campus (Murcia o Cartagena) y sala a la que deseas acceder.

 
y sala a la que deseas acceder.

 
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.4. Selecciona la fecha y la franja horaria disponible.

 
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.5. Selecciona tu asiento (fila y mesa). En la parte superior de la aplicación aparece un mapa orientativo donde puedes ver dónde se ubica tu puesto.
 
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.6. ¡IMPORTANTE! Cuando llegues a tu asiento, recuerda escanear el código QR que está en tu puesto (una sola vez). Si en los primeros 30 minutos no lo has escaneado o llegas tarde, automáticamente se cancelará tu reserva y se liberará tu puesto para que pueda reservarlo otro usuario.
SECTION: Aplicación de reserva de espacios y ¿Cómo reservar un asiento en la Biblioteca-Sala de estudio?
2.7. Si sales antes de acabar el turno, vuelve a escanear el QR de tu puesto para liberar tu sitio y pueda ser reservado por otra persona. Si has llegado al final del turno, no hace falta que lo vuelvas a escanear.