eamag/protocols-with-steps huggingface dataset:
TITLE: Standard CD Measurements
AUTHORS: Rohanah  Hussain, Charlotte S. Hughes, Giuliano  Siligardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.</div><br/><div class = "text-block">This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.</div><br/><div class = "text-block">The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules tomeasure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.</div></div>

[STEPS]
?. See 'Guidelines' section.
TITLE: Carbon and nitrogen stable isotope analysis in harbor seal pup fur at CICESE
AUTHORS: Juan Carlos Herguera-García, Gisela Heckel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In the Stable Isotope Laboratory at CICESE (Ensenada Center for Scientific Research and Graduate Education, Mexico) animal tissues (pup fur from Pacific harbor seals, </span><span style = "font-style:italic;">Phoca vitulina richardii</span><span>)  were ground for isotopic determinations. Samples were stored in a dissecator until they were loaded into a Costech</span><span style = "vertical-align:super;">®</span><span> Carrousel. Samples were combusted at high temperature (1000˚C) in a pure oxygen atmosphere in an elemental analyzer coupled to a Finnigan MAT Delta V Advantage continuous flow stable isotope mass spectrometer.</span></div><div class = "text-block"><span>Stable carbon and nitrogen ratios are expressed as δ</span><span style = "vertical-align:super;">13</span><span>C or δ</span><span style = "vertical-align:super;">15</span><span>N according to the following equation:</span></div><div class = "text-block"><span style = "vertical-align:super;">δ</span><span style = "vertical-align:super;">13</span><span>C or δ</span><span style = "vertical-align:super;">15</span><span>N (‰) = [Rsample/Rstandard)–1]* 1000 (1) </span></div><div class = "text-block"><span>where R is </span><span style = "vertical-align:super;">13</span><span>C/ </span><span style = "vertical-align:super;">12</span><span>C or </span><span style = "vertical-align:super;">15</span><span>N/ </span><span style = "vertical-align:super;">14</span><span>N. </span></div><div class = "text-block">Isotopic values of carbon and nitrogen are reported relative to Pee Dee Belemnite and atmospheric nitrogen standards, respectively. The accuracy of isotopic measurements was verified using secondary standard reference materials (Glutamic acid, a pure carbonate Merck and the laboratory internal references Lanugo for N, and CH94 for C).</div></div>

[STEPS]
TITLE: IMS Data Processing
AUTHORS: Lukasz Migas, Angela R.S.  Kruse, Jamie Allen, ellie.l.pingry, Melissa Farrow, Jeff Spraggins, Raf Van De Plas

[DESCRIPTION]
This protocol describes spectral alignment and mass calibration of IMS data beginning with Bruker (.d) raw files. Mass calibration is performed based on several well-characterized lipids. 

[STEPS]
1. Extract the content of the Bruker (.d) raw file in pseudo-profile mode and at native spectral resolution into memory or into a format you can calculate within your compute environment of choice. If multiple regions of interest were acquired in the same experiment/file, split these into separate datasets to simplify subsequent analysis.
2. Perform spectral alignment (alignment along the m/z axis) on each mass spectrum to bring it in line with the m/z axes of the other spectra in the dataset. 

During the data acquisition, minor changes to the instrument conditions might affect the alignment of peaks in the mass spectra, resulting in small mass or m/z deviations that need to be corrected. 
2.1. Spectral alignment is carried out using the Python msalign package (https://github.com/lukasz-migas/msalign).
2.2. Alignment aims to correct systematic shifts along the spectral domain, hence, we automatically select between 6 and 10 peaks from the mass spectrum and perform a linear alignment.
2.3. The automatically selected peaks should be present in the majority of the spectra since they act as anchors from which msalign estimates correction factors. Alternatively, it's possible to specify theoretical masses, in which case msalign would perform alignment and calibration in one step.
 

3. Create an average mass spectrum of all pixels in the dataset and use it as a basis for mass calibration. Several well-characterized lipids can be used for calibration purposes:

    Positive mode: m/z 703.575, 732.553, 734.569, 758.569, 760.585, and 782.567
    Negative mode: m/z 687.545, 714.508, 722.513, 744.555, 766.539, and 885.549
4. Perform mass calibration by fitting multiple linear regression models and using RANdom SAmple Consensus (RANSAC) to randomly resample the list of mass calibrants for each model. Select the calibration model that results in the smallest average ppm error across all calibrant species.
5. Calculate total ion current (TIC) normalization factors for each pixel (post-alignment and post-calibration if performed).
6. Perform m/z feature selection and annotation. See protocol below:
 

7. For each selected and annotated feature, extract its peak intensity from the mass-aligned, mass-calibrated dataset. 
 

8. Each ion image is ion intensity normalized using the TIC normalization factors calculated previously.
9. Export the dataset to an imzML file format using the Python pyimzml library (https://github.com/lukasz-migas/pyimzML).
TITLE:     ELISA for quantification of IL-15 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Monocytes, epithelium, and muscles make IL-15. It acts on T cells and activated B cells. It causes the proliferation of both B and T cells. It causes NK cell memory and CD8+ T cell proliferation. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-15 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-15 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-15 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-15 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-15 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-15 standard dilutions and the human IL-15 sample concentration is determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In this protocol we publish the maps and sequences of specific plasmids designed for the transformation of Bigelowiella natans, Pyramimonas parkeae, Eutreptiella gymnastica, Chromera velia and Pseudonitzchia delicatissima. All these plasmids contain gene for firefly lucipherase as a reporter, some of them contain PAC (puromycin) or G418 (neomycin) resistance gene. All genes are flanked by organism specific up- and downstream gene regions extracted from genomic and transcriptomic data. It should be noted that we were not successful in any transformation by these plasmids, but it does not mean that the problem is in the plasmids. All plasmids are available upon request. The plasmid maps and sequences can be downloaded as a word file from More/Material section of this protocol. </p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
M2 medium for co-culturing Cyanobacteria, such as S. elongtatus, together with heterotrophic bacteria


Slighly modified from: 
Weiss, Taylor L., Eric J. Young, and Daniel C. Ducat. "A synthetic,  light-driven consortium of cyanobacteria and heterotrophic bacteria  enables stable polyhydroxybutyrate production." Metabolic engineering 44 (2017): 236-245.

[STEPS]
SECTION: Ingredients
?. 
SECTION: Ingredients
?. 
SECTION: Ingredients
?. 
SECTION: Ingredients
?. 
SECTION: Trace metal mix
?. 
SECTION: Ingredients
?. 
SECTION: Ingredients
?. 
SECTION: Ingredients
?. 
SECTION: Important information
?. 
SECTION: After autoclaving
?. 
SECTION: After autoclaving
?.
TITLE: Rat brain processing for histological analyses (update)
AUTHORS: miquel.vila

[DESCRIPTION]
Protocol for rat brain processing in order to perform histological analyses

[STEPS]
SECTION: Rat perfusion
1. Deeply anesthetize animals with sodium pentobarbital (50 mg/kg, i.p.)
SECTION: Rat perfusion
2. Perfuse through the left ventricle with saline [0.9% (wt/vol)] at room temperature (RT)
SECTION: Rat perfusion
3. Perfuse again with ice-cold formaldehyde solution 4% in PBS buffered for histology

SECTION: Postfixation
4. Remove brains and post-fix them for 24 h in the same fixative
SECTION: Processing for microtome sectioning
5. Wash twice with 0.1 M PBS and process for paraffin embedding following standard procedures (performed by an external facility) 

Alternatively, brains could be stored in 0.1 M PBS at 4 °C (not for over a month without changing the PBS) prior paraffin processing
SECTION: Processing for cryostat sectioning
6. Cryoprotect for 24-48 h (until they sink) in 30% sucrose at 4 °C
SECTION: Sectioning
10. Perform sectioning with a sliding microtome at 5-μm-thickness for paraffin samples or
in a cryostat at 20- or 30-μm-thickness for frozen samples. 
SECTION: Processing for cryostat sectioning
7. Exchange sucrose for 0.1 M PBS
SECTION: Processing for cryostat sectioning
8. Immerse brains in cold (-30ºC) 2-methylbutane for 30 s and store at -80 °C
SECTION: Processing for cryostat sectioning
9. Include in OCT
TITLE: Immunoflourescent assay
AUTHORS: Binnypreet Kaur1, 2 ,  Drahomíra Faktorová1, 2,  Priscila Peña-Diaz1 and Julius Lukeš1, 2

[STEPS]
?. Store it in dark at 4°C until use
?. Preferably night before, make 4% paraformaldehyde (w/v) in PBS and incubate O/N at 37°C
?. 
?. Spin down the cells 3000RPM for 2min and discard the supernatant in formaldehyde waste
?. Mix the pellet with 500ul of fixer in the hood and incubate the slides for 10min in humid dark chamber at room temperature
?. Remove excess of the paraformaldehyde  by tilting the slide on the paper
?. Permeabilization with chilled methanol for 30min than 0.2% 1XPBS triton for 20min in dark
?. Slightly wash the cells with 1x PBS to remove detergent and make it air dry
?. Incubate the permeabilized cells with 5%milk prepared with 1X PBS+0.05% Tween 20 for 30min to 2h
?. Incubate the cells with primary antibody diluted in 5%milk (1X PBS+0.05% Tween 20) at 4°C
?. Wash the slides 3times with 1X PBS before adding secondary antibody diluted in 1% BSA (w/v) and incubate for 1h
?. Wash the slides 3 times in 1X PBS for 5min and tap away the  moisture, to let it air dry
?. Drop on each sample 1 drop of Prolong Gold Antifade with DAPI(Invitrogen P36935)
?. Mount the slide with coverslip and sail with nail polish
?. Let it dry in the humid chamber for 10 min and can store in dark at 4°C until observing under the microscope.
?. Wash the slides 3times with 1X PBS before adding secondary antibody diluted in 1% BSA (w/v) and incubate for 1h. After incubation washes the slides 3X times with 1X PBS. Make it air dry.
?. Add one drop of Prolong Gold Antifade with DAPI(Invitrogen P36935) Mount the slide with the coverslip and sail with nail polish.
?. Let it dry in humid chamber for 10min and so on.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This Real-Time PCR genotyping protocol can be used for the detection of polymorphisms at codons 146 (N/S/D), 211 (R/Q) and 222 (Q/K) in the caprine <em>PRNP</em> gene. We developed four seperate Real-Time PCR reactions with four different Custom TaqMan<sup>® </sup>SNP Genotyping Assays (Applied Biosystems, Foster City, California, USA). Each SNP Genotyping Assay consisted of a mix of sequence-specific forward and reverse primers to amplify the polymorphic sequence of interest and two TaqMan<sup>® </sup>MGB probes, FAM and VIC dye-labeled to detect the amplified product.</p>

[BEFORE_START]
<p>Always ensure that the Genotyping Assays (primers and probes) have been fully thawed and mixed before use.</p>

[GUIDELINES]
<ul>
<li>Genotyping Assays (primers and probes) should be stored protected from light and at -20 <sup>o</sup>C when not in use. Do not perform more than 10 freeze-thaw cycles.</li>
<li>KAPA PROBE FAST qPCR Master Mix should be stored at -20 <sup>o</sup>C when not in use. Do not perform more than 30 freeze-thaw cycles.</li>
<li>Avoid repeated freezing and thawing of all reagents.</li>
</ul>

[STEPS]
?. 
?. 
?.
TITLE: Mammalian Cell Staining
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to stain mammalian cells cultured on a 96-well plate. Actin filaments, focal adhesion sites (as indicated by the presence of vinculin), and nuclei will be stained.</div></div>

[STEPS]
?. [Fix the cells]
Remove cell culture media.
You may wash twice with DPBS at this point.
?. [Perforate cell membrane]
Remove paraformaldehyde solution.
?. [Block unspecific binding]
Remove Triton X-100.
?. [Stain actin filaments and focal adhesion sites]
Add FITC:TRITC solution
100 µl
?. [Bind anti-vinculin to vinculin]
Add of Anti-Vinculin and blocking buffer mixture.
100 µl
?. [Fix the cells]
Add  of  paraformaldehyde solution.
100 µl
4 Volume Percent
?. [Fix the cells]
Incubate for .
?. [Fix the cells]
Transfer flask to CO2 incubator.
?. [Fix the cells]
Label the flask with:Cell linePassage numberDateYour intials
?. [Perforate cell membrane]
Incubate for .
?. [Perforate cell membrane]
Add of Triton X-100.
100 µl
0 Volume Percent
?. [Perforate cell membrane]
Wash twice with  washing buffer.
100 µl
?. [Block unspecific binding]
Incubate for.
?. [Block unspecific binding]
Add blocking buffer.
100 µl
?. [Block unspecific binding]
Wash twice with washing buffer.
?. [Bind anti-vinculin to vinculin]
Incubate for .
Increasing this duration can greatly increase FITC-conjugated secondary antibody binding. It is acceptable to place covered plate in refrigerator overnight. While waiting on this incubation, it is a good time to prepare the combined dye solution.
?. [Bind anti-vinculin to vinculin]
Remove blocking buffer.
?. [Bind anti-vinculin to vinculin]
Wash twice with washing buffer.
?. [Stain actin filaments and focal adhesion sites]
Incubate for .
?. [Stain nucleus]
Remove stains.
?. [Stain nucleus]
Add of DAPI solution
100 µl
?. [Stain nucleus]
Wash twice with washing buffer.
?. [Stain nucleus]
Incubate for
?. [Stain nucleus]
Remove DAPI.
?. [Stain nucleus]
Add PBS to prevent dessication of cells during imaging/storage.
100 µl
?. [Stain actin filaments and focal adhesion sites]
Remove anti-vinculin blocking buffer mixture.
?. [Stain actin filaments and focal adhesion sites]
Wash twice.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The protein encoded by this gene is proteolytically processed to release a secreted peptide termed endothelin 1. This peptide is a potent vasoconstrictor and is produced by vascular endothelial cells. Endothelin 1 also can affect the central nervous system. Two transcript variants encoding different isoforms have been found for this gene.</p>

[STEPS]
?.
TITLE: Catalepsy test in rats
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing the catalepsy bar test in rats. This test evaluates muscular rigidity, and can be used in rat models with motor deficits, such as models of Parkison's disease.

[STEPS]
SECTION: Test
1. Bring cages to the behavioral room for at least 60 min prior to the test for habituation

SECTION: Test
2. Place both forepaws of the rat on an horizontal bar elevated so that the rat is in an half rearing position
SECTION: Test
3. Measure the descent latency, defined as the time it takes for both forepaws to be removed from the bar
SECTION: Test
4. Repeat the test three times, with a brief rest in between
TITLE: KAPP-Sen TMC: Dissociation of Pancreatic Islets (recovered)
AUTHORS: Juliana Alcoforado Diniz, Jessica Garofalo, Dylan Baker, Paul Robson

[DESCRIPTION]
The dispersed samples were shipped cold from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets were recovered and dissociated as follows.

[STEPS]
SECTION: Cell Dissociation with Accutase
1. 
 

SECTION: Fixation of Cells &amp; Nuclei for Chromium Fixed RNA Profiling
2. Cells were fixated prior to scRNAseq according to https://dx.doi.org/10.17504/protocols.io.x54v9py5zg3e/v1
SECTION: Cell Dissociation with Accutase
1.1. Transfer cell suspension of pure islets to a new 50ml tube. Use additional media to rinse original container.
SECTION: Cell Dissociation with Accutase
1.2. Centrifuge at room temperature 130g for 5 mins.
SECTION: Cell Dissociation with Accutase
1.3. Aspirate the supernatant and add media to the appropriate concentration of 1,000 islets/1 ml.
--> Ex: If receiving 7,000 islets, use 7 ml media.
SECTION: Cell Dissociation with Accutase
1.4. To recover cells, add half of the islets to a coated flask and incubate at 37°C overnight.
SECTION: Cell Dissociation with Accutase
1.5. Coat pipette and add islets from flask to a 50ml tube. Wash flask with media to make sure all islets have been collected.
SECTION: Cell Dissociation with Accutase
1.6. Centrifuge at 130 g, room temperature for 2 mins.
SECTION: Cell Dissociation with Accutase
1.7. Aspirate media and resuspend in 4 ml accutase. Incubate at 37C for 8 mins, mixing with pipette every 2 mins. Check at 6 mins.                                                                                                                                      -->1 mL accutase/1,000 islets
SECTION: Cell Dissociation with Accutase
1.8. Add CMRL 1066 (Cat. 11530037) media to 25 ml then centrifuge at 230 g.
SECTION: Cell Dissociation with Accutase
1.9. Aspirate supernatant and resuspend in 1.5 ml of CMRL
SECTION: Cell Dissociation with Accutase
1.10. Filter through a 40 µm Flowmi.
SECTION: Cell Dissociation with Accutase
1.11. Count cells using AO/PI (acridine orage/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
SECTION: Cell Dissociation with Accutase
1.12. Proceed to cell fixation.