eamag/protocols-with-steps huggingface dataset:
TITLE: B-PER Lysis--CHEM 384
AUTHORS: Ken  Christensen

[DESCRIPTION]
The Thermo Scientific B-PER Bacterial Protein Extraction Reagent enables mild extraction of proteins from bacteria (E. coli) without the need for mechanical disruption. The reagent may be used for soluble protein extraction and inclusion body purification from bacterial cell lysates. The B-PER Reagent with Enzymes is supplied with lysozyme and DNase I to improve the extraction efficiency of large (> 70 kDa) molecular weight proteins and proteins expressed in inclusion bodies.

[GUIDELINES]
Important Product Information

The B-PER Reagent extracts proteins from recently prepared cells and frozen cells. Extraction is typically more effective with frozen cells. The amount of reagent required depends on the amount of cell paste.

Important Product Information • The B-PER Reagent extracts proteins from recently prepared cells and frozen cells. Extraction is typically more effective with frozen cells. The amount of reagent required depends on the amount of cell paste.

B-PER Reagent effectively extracts soluble proteins from several common bacterial host strains and is especially suitable for the protease-defective expression host BL21 strains. If lysis is inefficient for a particular bacterial strain, freeze cells before extraction. 

[STEPS]
SECTION: B-PER Lysis to extract soluble protein
1. Pellet bacterial cells by centrifugation at 5000 × g in a tared tube for 10 min.

SECTION: B-PER Lysis to extract soluble protein
5. Optional: Add 2 µLof lysozyme and 2 µL of DNase I per 1 mL of B-PER Reagent added.

SECTION: B-PER Lysis to extract soluble protein
4. Add 4 mL of B-PER Reagent per gram of wet cell pellet. Pipette the suspension up and down until it is homogeneous.

SECTION: B-PER Lysis to extract soluble protein
6. Incubate 15 min at room temperature.

SECTION: B-PER Lysis to extract soluble protein
7. Centrifuge lysate at  high speed (~15,000 × g) for 5 min to separate soluble proteins from the insoluble proteins. 
 


SECTION: B-PER Lysis to extract soluble protein
2. Decant your spent LB and determine the mass of your cell pellet.
SECTION: B-PER Lysis to extract soluble protein
3. Freeze your pellet in the -20°C freezer overnight or quickly freeze your pellet in liquid nitrogen. When you are ready to proceed, thaw your cells at room temperature.
TITLE: Nextera XT at 0.2X On the Mantis
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to generate Nextera libraries using 0.2x reagents in a 96-well PCR plate using the Formulatrix Mantis instrument.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Foulage test
AUTHORS: Tomohisa  Yasuda, Toru Miwa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Foulage test is a test in which the stepping is recorded with the eyes open and closed by the ground contact method for 60 seconds in accordance with the metronome BPM120 by counting the sway of the center of gravity. The enlargement of the area of the still-standing object on a stabilometer means unstable directly. However, the dynamic test must divide the unstable movement to the active. The stepping of Foulage test is regulated that 120bpm tempo, upright standing with both arms set on the body-side, foot closed, toes keep touching on the plate constantly, so object can change only height of heels to rise up alternatively. Foulage is a French word for "grape trampling" in which grapes are crushed barefoot in the process of winemaking. This is because the ground contact method is different from the conventional stepping method of a march that raises the sole of the foot.</div><div class = "text-block">In a stable stepping of a healthy person, it is considered that the left and right folds and the front-back shift are constant to some extent, and 120 steps and 60 reciprocations are added to the same locus, and an inverted V-shaped locus with a certain width. Here, the inverted V-shaped locus is close to a rectangle bent at the center, and its long side can be considered as the average moving distance (L / 120) of one step. The outer peripheral area of ​​the inverted V-shaped locus, that is, the area A of the bent rectangle can be estimated by multiplying the length of the long side and the short side (front-back width). We defined front-back width as FT value. </div><div class = "text-block">Our results in normal subjects showed that the height of heels enlarges the total locus length. Pearson correlation analysis revealed that FT value of healthy object plateaued under stable condition (400<L<1400). Therefore, the test should be performed within the range of heel height where the footsteps are stable in healthy subjects. In addition, we proposed the new parameters; the variance of steps is more direct parameter of stability. The plotting figure of steps and the time-graph can show the movement of a subject visually.</div></div>

[STEPS]
?. 1st: A step on the center of stabilometer (GP5000, Anima, Tokyo, Japan) with foot closed, lifting only the heels alternately with the metronome at a tempo of 120 BPM, for 60 seconds with eyes open.
?. 2nd: A step on the center of stabilometer (GP5000, Anima, Tokyo, Japan) with foot closed, lifting only the heels alternately with the metronome at a tempo of 120 BPM, for 60 seconds with eyes closed.
?. 3rd: FT value is automatically calculated, and graphs are drawn in the Foulage test software. The variance of steps is manually calculated, and graphs are drawn in the Excel file.
TITLE: Protocol S-100B Determination in Melanoma
AUTHORS: Samantha Damude, Schelto  Kruijff, Harald  J Hoekstra, Kevin  P Wevers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the implementation of a dummytube system in AJCC stage III melanoma patients, to investigate whether serum S-100B levels can be falsely elevated by adipocyte contamination in the first drawn serum separation tube. </div><div class = "text-block">A dummy tube system was introduced for performance of venipunctures during regular follow-up in all AJCC stage III melanoma patients, with no evidence of disease.After venipuncture, a dummy tube was drawn first, and subsequently the regular tube was drawn. The first tube was anonymously coded and stored, while the second tube was registered in the patients’ medical results. After performing the assay, S-100B levels between the two consecutively drawn samples were compared.</div></div>

[STEPS]
TITLE: Cell culture, transfection, and imaging of K562 cells
AUTHORS: Chase Amos, Pietro De Camilli

[DESCRIPTION]
This protocol details the general preparation of K562 cells for imaging.

[STEPS]
SECTION: Cell culture
1. Culture K562 cells at 37 °C  and 5% CO2 in RPMI containing 10% FBS, 1 millimolar (mM) sodium pyruvate, 100 U/mL penicillin, 100 μg/mL  streptomycin, 2 millimolar (mM)  L-glutamine, 1 x  non-essential amino acids, (all from Gibco) and 2.5 μg/mL plasmocin (InvivoGen).
 

SECTION: Transfection and imaging
2. For imaging experiments, seed the cells on fibronectin (Sigma Aldrich) 35mm imaging dishes (MatTek) at a concentration of 2x105 cells per dish in RPMI without antibiotics. Allow cells to attach 960 min  at 37 °C  and 5% CO2.

SECTION: Transfection and imaging
3. After cells attach, replace media with RPMI supplemented with hemin (Sigma Aldrich) dissolved in DMSO to a final concentration of  30 micromolar (µM). Transfect transiently with plasmids by adding FuGene 4K (Promega) and incubate   . After the overnight incubation, replace the media with new media containing the same factors of hemin and (where applicable) transfection reagent and plasmids, and incubate   .

SECTION: Transfection and imaging
4. Following the 2 days of transfection/hemin treatment, prepare the cells for imaging. Where applicable, add Halo ligand and incubate for 90 min  at 37 °C , 5% CO2. Replace with new RPMI media (supplemented at 30 micromolar (µM) hemin if differentiating cells) prior to imaging. 

If using TMRE (Cayman Chemical, TMRE Mitochondrial Membrane Potential Assay Kit), add prior to imaging at a final concentration of 200 nanomolar (nM)  TMRE.

SECTION: Transfection and imaging
5. Perform spinning-disk confocal microscopy using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera. Cells are imaged at 37 °C  and 5% CO2.
SECTION: Transfection and imaging
6. Identify the cells to be imaged by scanning the dish.
TITLE: DNA Concentration Measurement (Protocol for Thermo Scientific NanoDrop™ 1000 Spectrophotometer)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol adapted from the NanoDrop 1000 Spectrophotometer V3.8 User’s Manual. </div></div>

[STEPS]
?. Open the ND-1000 V3.8.1 Program using the computer (the computer should be connected to the Nanodrop apparatus).
?. Click on the corresponding application module (e.g., Nucleic acid to determine the concentration and purity of nucleic acid).
?. Open the sampling arm and load a blank sample (e.g., TE Buffer, MQ Water, etc.).
1.5 µl
?. Close the sampling arm on the machine to cover the blank sample.
?. Click "OK" to read the blank.
?. Open the sampling arm and clean the blank off the upper and lower pedestals using a Kim Wipe.
?. Load your DNA or RNA sample and close the sampling arm.
1.5 µl
?. Select the sample type in the program (e.g., DNA or RNA).
?. Click "Measure".
?. When finalized, read the 260/280, 260/230 and concentration (ng/µL) measurements.
?. Clean the nucleic acid sample off of the upper and lower pedestals using a Kim Wipe.
?. Close program.
?. Repeat for all samples.
TITLE: Methanol Precipitation of Proteins
AUTHORS: Tobias von der Haar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is adapted from </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/6731838" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Wessel and Flugge, 1984</span></a><span>. It is suitable for precipitating very dilute proteins, and proteins from solutions containing detergents.</span></div></div>

[STEPS]
?. Select a suitable container for the precipitation. This protocol requires the addition of large amounts of additional liquid to the original sample, so the container needs to be sufficiently large as well as suitable for centrifugation. Maximal sample sizes for the following container volumes are (tube volume: maximal sample volume): 1.5 ml: 0.16 ml; 2 ml: 0.22 ml; 10 ml: 1.1 ml; 15 ml: 1.6 ml; 50 ml 5.5 ml.
?. Place your sample in the selected container. Note the sample volume, and use this to calculate the amount of reagents to add in subsequent steps.
?. Followig the centrifugation you should observe two phases, an upper aqueous phase and a lower solvent phase. The protein is located at the interface between the phases (it may be visible as a white film, or it may not be visible at all if the protein amount is small).Pipet away and discard the upper, aqueous phase, taking care not to disturb the interface (it is ok to leave some of the aqueous phase behind).
?. Add 3 volumes of methanol to the tube, vortex, and centrifuge at 9000 g, 4°C for ten minutes.
?. Mix the sample with 4 volumes of methanol. Vortex, then add 1 volume of chloroform and vortex again. Add 3 volumes of water, vortex, and centrifuge at 9000 g, 4°C for five minutes.
?. Pour off the liquid  and dry the pellet by standing the tube, open, upside down on a clean, dry tissue. Dissolve the pellet in an appropriate volume of buffer (eg SDS sample buffer if the precipitate is to be analyses by SDS PAGE).
TITLE: Frequentist, Bayesian Analysis and Complementary Statistical Tools for Geriatric and Rehabilitation Fields: Are Traditional Null-hypothesis Significance Testing Methods Sufficient?
AUTHORS: Dahan da Cunha Nascimento

[DESCRIPTION]
Null hypothesis significant testing (NHST) is the dominant statistical approach in the geriatric and rehabilitation fields. However, NHST is routinely misunderstood or misused. In this case, the findings from clinical trials would be taken as evidence of no effect, when in fact, a clinically relevant question may have a "non-significant" p-value. Conversely, findings are considered clinically relevant when a significant difference is observed between groups. To assume that p-value is not an exclusive indicator of an association or the existence of an effect, researchers should be encouraged to report other statistical analysis approaches as Bayesian analysis and complementary statistical tools alongside the p-value (e.g., effect size, confidence intervals, minimal clinically important difference, and magnitude-based inference) to improve interpretation of the findings of clinical trials by presenting a more efficient and comprehensive analysis. However, the focus on Bayesian analysis and secondary statistical analyses does not mean that NHST is less important. Only that, to observe a real intervention effect, researchers should use a combination of secondary statistical analyses in conjunction with NHST or Bayesian statistical analysis to reveal what p-values cannot show. This paper provides potential insights for improving scientific data interpretation in rehabilitation and geriatric fields by utilizing Bayesian and secondary statistical analyses to better scrutinize results of clinical trials where a p-value alone may not be appropriate to determine efficacy of an intervention.

[STEPS]
SECTION: Supplementary materials
1.
TITLE: Installing FIJI and SynBot (Windows Version)
AUTHORS: Justin T Savage

[DESCRIPTION]
Video instructions for installing FIJI and SynBot and a simple SynBot run for Windows operating system. 

[STEPS]
1. 
 

SECTION: Install FIJI
2. 0:00 - 0:40 Install FIJI from https://fiji.sc .
SECTION: Install SynBot
3. 0:40 - 1:37  Download code from the SynBot GitHub repository https://github.com/Eroglu-Lab/Syn_Bot .
SECTION: Install SynBot
4. 1:37 - 2:09  Move SynBot java plugins (JAR files) to the FIJI plugins folder. The ilastik4ij_Syn_Bot-1.8.2-SNAPSHOT.jar and SynQuantExtra-1.2.9.jar files are required for running SynBot.
SECTION: Run SynBot
5. 2:09 - 2:22 Launch FIJI and open SynBot by dragging the SynBot.ijm file into FIJI.
SECTION: Run SynBot
6. 2:22 - 2:49 Prepare your image folder. Create a folder for your experiment with subfolders for experimental groups. Each experimental group folder should contain only image files. Multiple experimental groups are not required, but your files must follow the Experiment/Group/Image structure. 
SECTION: Run SynBot
7. 2:49 - 3:41 Run SynBot (see the SynBot manual thresholding protocol for more information).
SECTION: Run SynBot
8. 3:41 - 4:12 View results in Summary.csv file.
1.1. If video quality is poor, try watching directly from Youtube at https://youtu.be/VZqD8DS_VfA?si=DFcoeyiLyKIuYUUX
TITLE: The impact of globalization on oral health in the Pacific
AUTHORS: Kenechi Elvis Obiorah

[DESCRIPTION]
    A literature and art review of oral health at the time of European contact and throughout written history and a survey on the oral health quality of native Pacific Islanders today to examine the effects of globalization more specifically the introduction of highly processed carbohydrates and sugars have influenced the oral health of Indigenous Pacific Islanders.

[STEPS]
SECTION: Oral health quality and hygiene practices of native Pacific Islanders on Moorea, French Polynesia
1. Art pieces showing oral health quality (full set of teeth) will be compiled into a digital document. A literature review of journals and papers describing oral health quality from missionary/colonists/military accounts during the early contact period in the Pacific will be compiled aswell. Information on pre-contact forms of oral health hygiene practices will also be compiled.
SECTION: Oral health quality and hygiene practices of native Pacific Islanders on Moorea, French Polynesia
2. Older native Pacific Islander participants will be surveyed additionally about the changes they've seen in terms of oral hygiene practices and oral health to further examine globalization's effects on oral health.
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
3. On a "food-for-thought truck" data will be collected during dental consultations by Tahitian dentists and compiled to access the oral health quality of each patient. Cavities, gum diseases, severe tooth decay/dead teeth, and gum recession are the data points. Each oral health disease will be ranked from 1 to 5. This data will be scaled to a standard oral health quality expected for each participant's age range.
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
4. Diet information will also be collected, and separated into two major categories, a more traditional diet vs a more "Western diet". Of both diets, food types are to be accessed, foods high in refined sugar, high calcium/vitamin D foods, and foods rich in fluoride.
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
5. Then oral hygiene practices will be surveyed, the western standard is to brush at least twice a day and floss at least once. Other oral hygiene practices will be surveyed aswell: the use of chewing sticks, the use of picks, and the use of salt which have all been proven beneficial for oral health.
TITLE: VLP Extraction from Fecal Samples
AUTHORS: Frej   Larsen

[DESCRIPTION]
This protocol is for isolation of bacteriophages from fecal matter. It is based on the protocol used for the COPSAC10 cohort and was originally described by Ling Deng in doi:10.3390/v11070667. Extraction is performed using centrifugation and ultrafiltration to isolate viral particles and get rid of contaminants.

[BEFORE_START]
Fill bucket with crushed ice

Turn on centrifuge and start the fast temp program to reach 4◦ C

Cool SM buffer in ice bucket

[GUIDELINES]
This protocol is designed for extraction of bacteriophages from fecal samples. The sample should be 50-500mg of fecal material diluted in buffer (2xSM buffer for instance). Sample sizes outside these limits have not been tested and correct sequencing results can not be guaranteed.

[STEPS]
1. Thaw frozen sample in ice bucket
2. Transfer up to 500 mg of sample to centrifuge tube and add SM buffer to reach a final volume of 5 mL. Place centrifuge tube back in ice box

3. Homogenize samples by placing ice box on shaking board at   

4. Centrifuge samples at5000 rcf, 30 min, 4 °C 

5. Filter the supernatant  through a 0.45 um PES filter into a new centrifuge tube
6. Transfer 2.5 mL of the sample to the outer chamber of the concentrator

7. Centrifuge sample in the concentrator at 1500 rcf, 20 min, 4 °C 

8. Pour off liquid from the inner chamber
9. Repeat the three previous steps until all liquid from the centrifuge tube has passed through the concentrator and the there is no liquid left in the inner chamber
10. Add 100 ul SM buffer to the inner chamber and centrifuge 1500 rcf, 2 min, 4 °C 

11. Leave in fridge to release VLPs from filter for at least 12 hours and at most for 1 week before proceeding with spotting or DNA/RNA extraction
TITLE: Immunohistochemistry on free-floating cryosections
AUTHORS: Nuriapenuelas

[DESCRIPTION]
Immunochemistry protocol on rodent brain cryosections 

[STEPS]
SECTION: 1. Section selection
1. Collect the cryosections needed for a caudo-rostral representation of each brain region (every fourth section or every sixth section dependeing on the section thinknes, brain region and animal species) into 24-well-plate (3-4 sections per well).

SECTION: 2. Blocking endogenous peroxidase
2. Incubate sections in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol for 10 min (500uL/well)
SECTION: 3. Blocking
3. Incubate sections in blocking in TBS 1X + 5% NGS or NDS (500uL/well) 1h at RT
SECTION: 4. 1ary antibody
4. Incubate sections inTBS 1X + 2% NGS or NDS + primary Ab 24/72h (it depends of the Ab) at 4ºC (cold room).
SECTION: 5. 2ary antibody
5. Incubate sections in 2% NGS or NDS + Secondary Ab 1h at RT.
SECTION: 5. 2ary antibody
5.1. At this step it is important to prepare ABC solution and let it, at least, 30 min on the shaker
SECTION: 6. ABC incubation
6. Incubate 1 hour at RT with ABC solution (Ultra-Sensitive or Standard ABC Peroxidase Standard Staining Kit).
SECTION: 7. Developing
7. In aluminium foil: DAB Standard Kit (1 drop of reagent B in 1 mL of reagent A, gives rise to brown staining), or Vector SG (3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS, gives rise to blue staining).
8. Put 500 uL on each well and put a cardboard box on it to keep darkness for a time ranging from 3-15 minutes depending on the antibody used.
8.1. Remove with an air-pump equipment and clean the material with bleach.
SECTION: 9. Dehydratation
10. Incubate slides in consecutive ethanol solutions (1 min in ethanol 70%-1 min in ethanol 95%-1 min in ethanol 100%).
SECTION: 9. Dehydratation
11. Incubate slides in 2x5 min in Xylene.
SECTION: 10. Mount coverslips
12. Put a line of mounting medium (DPX) by slide. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles.
13. Let dry the slides in the hood overnight.
SECTION: 2. Blocking endogenous peroxidase
2.1. Wash 3x5 min in TBS 1X.
SECTION: 1. Section selection
1.1. Wash 3x5 min in TBS 1X. Put 500ul per well and aspirate the liquid with an air-pump equipment.
SECTION: 4. 1ary antibody
4.1. Wash 3x5min in TBS 1X.
SECTION: 5. 2ary antibody
5.2. Wash 3x5min in TBS 1X.
SECTION: 6. ABC incubation
6.1. Wash 3x5min in TBS 1X.
8.2. Wash 3x5min in TBS 1X. 
SECTION: 8. Mount sections
9. Mount sections into slides and let it dry overnight.