eamag/protocols-with-steps huggingface dataset:
TITLE: Assessment of TNF-alpha and  BDNF
AUTHORS: hananagm

[DESCRIPTION]
Abstract
Background: Cerebral palsy (CP) is the most common motor disability in children, which is instigated by damage to the developing brain that affects the ability to control the muscles. The main types of CP are spastic CP, dyskinesia CP and mixed CP. The aim of this work was to estimate the concentrations of complete blood count (CBC), erythrocytic sedimentation rate (ESR), C-reactive protein (CRP), brain-derived neurotrophic factor (BDNF), and tumor necrosis factor-α (TNF-α) in children with CP compared to the control group.
Methods: A total of 75 Egyptian children were enrolled in this study, 45 had CP and 30 were controls. CBC, ESR, CRP, BDNF, and TNF-α were assessed.
Results:The ESR, CRP and TNF-α levels showed statistically significant increases in cases compared with controls. While the neutrophil/lymphocyte ratio and the BDNF levels were significantly lower in CP compared with the controls. When comparing the different groups of CP with each other; there were no significant differences. Regarding the correlation of BDNF and different studied parameters, our study showed a positive correlation between BDNF and TNF levels only within the group with spastic CP.
Conclusions: BDNF may be considered as a biomarker or treatment target for CP to avoid further complications as still there is insufficient progress in the prediction, early diagnosis, treatment, and prevention of CP. Furthermore, searching for novel strategies to increase BDNF levels may open a new opportunity for the treatment of CP. 

[STEPS]
SECTION: Assessment of TNF-alpha and BDNF
1.  Catalog No ab181421      (www.abcam.com/ab181421      , BDNF ab212166  www.abcam.com/ab212166)

First separate the serum
SECTION: Assessment of TNF-alpha and BDNF
2. store samples in low degree properly  -10 c
SECTION: Antibody Cocktail
3. 300 ul of l HumanCapture Antibody + 300 ul of Detector Antibody + with 2.4 mL Antibody Diluent
SECTION: standard curve
4. serially diluted standards immediately
fresh set of positive controls for every use.
some was repeated in the well to check the accuracy
4.1. some was repeated in the well to check the accuracy
SECTION: ElISA  Steps
5. Add 50 μL of all sample or standard to appropriate wells.
SECTION: ElISA  Steps
6. Add 50 μL of the Antibody Cocktail to each well.
SECTION: ElISA  Steps
7. The plate was sealed and incubated for 1 hour at room temperature on a plate shaker set to 400 rpm
SECTION: ElISA  Steps
8. Wash each well with 3 x 350 µL Wash Buffer. Wash by aspirating or decanting from wells then dispensing 350 µL 1X Wash Buffer into each well. Complete removal of liquid at each step is essential for good performance. After the last wash invert the plate and blot it against clean paper towels to remove excess liquid
SECTION: ElISA  Steps
9. Add 100 µL of TMB Development Solution to each well and incubate for 10 minutes in the dark on a plate shaker set to 400 rpm
SECTION: ElISA  Steps
10. ADD 100 ul of stop solution to each well. shake plate on plate shaker for 1 min .
SECTION: ElISA  Steps
11. . Read the OD at 450 nm
TITLE: HisPur Purification of His-Tagged Proteins--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Thermo Scientific HisPur Ni-NTA Resin enables effective immobilized metal affinity chromatography (IMAC) purification of polyhistidine-tagged proteins from a soluble protein extract. This resin is composed of nickel-charged nitrilotriacetic acid (NTA) chelate immobilized onto 6% crosslinked agarose. The Ni-NTA resin is compatible with native or denaturing conditions and can be used in multiple formats, including conventional gravity-flow chromatography, spin column and FPLC. Ni-NTA resins are commonly chosen for His-tagged-protein purification because of the four metal-binding sites on the chelate, which allow for high-binding capacity and low-metal ion leaching.</div></div>

[STEPS]
?. Add  of a 50% Ni-NTA resinslurry to a 1.7 ml microcentrifuge tube. Centrifuge tube for  at 700 × g and carefully remove and discard the supernatant.
250 µl
The HisPur Ni-NTA Resin allows for purification strategy customization. Purification conditions can be scaled as needed. The procedure may be performed at room temperature or at 4°C.It is important to spin the resin only at speeds up to 700 x gYou should only use a 1 ml pipette tip when manipulating the resin.
?. Add two resin-bed volumes of Equilibration Buffer and mix gently until the resin is fully resuspended
Since you added   of the 50% resin slurry to your tube, your resin bed volume is
?. Centrifuge tube for  at 700 × g and carefully remove and discard buffer.
?. Prepare sample by mixing the protein extract from your B-PER Lysis with an equal volume of Equilibration Buffer. The total volume should equal at least two volumes of the resin bed.
?. Add the prepared protein extract to the tube and mix on an end-over-end rotator for 30 minutes.
?. Centrifuge the tube for  at 700 × g. If desired, save supernatant for downstream analysis.
?. Wash the resin with two resin-bed volumes of Wash Buffer. Centrifuge the tube for  at 700 × g. Save supernatant for downstream analysis.
?. Repeat wash step. Optional: You can monitor the washing by measuring the absorbance of the supernatant by  at 280 nm until a baseline is reached.
?. Elute bound His-tagged proteins using one resin-bed volume of Elution Buffer. Centrifuge tube for  at 700 × g. Carefully remove and save the supernatant. Repeat this step twice, saving each supernatant fraction in a separate tube.
?. Monitor protein elution by measuring the absorbance of the fractions at 280nm. Since you are purifying a fluorescent protein, you can also monitor the protein concentration by measuring the absorbance at 500 nm.
The eluted protein can be directly analyzed by SDS-PAGE. Samples containing 6M guanidine•HCl must be dialyzed against a buffer containing 8M urea before SDS-PAGE analysis.
TITLE:        ELISA for quantification of granulocyte macrophage-colony stimulating factor (GM-CSF) in tissue culture supernatant, human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Granulocyte macrophage-colony stimulating factor (GM-CSF) is a monomeric glycoprotein. It is a cytokine secreted by macrophages, T cells, natural killer cells, mast cells, endothelial cells and fibroblasts. It acts as a growth factor. [1] </div><div class = "text-block">Reference</div><div class = "text-block">1. Egea L, Hirata Y, Kagnoff MF. GM-CSF: a role in immune and inflammatory reactions in the intestine. Expert Rev Gastroenterol Hepatol. 2010 Dec;4(6):723-31. doi: 10.1586/egh.10.73. PMID: 21108592; PMCID: PMC3291482.</div></div>

[STEPS]
?. An anti-human granulocyte macrophage-colony stimulating factor (GM-CSF) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma into the wells. GM-CSF present in the serum sample binds to antibodies adsorbed into 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-GM-CSF 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-GM-CSF antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of GM-CSF present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human GM-CSF standard dilutions and the human GM-CSF sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the correct protocol if you are using the C2987I cells. If you are using the C2987H cells, please refer to <a href="http://protocols.io/view/High-Efficiency-Transformation-Protocol-C2987H-imst3v" target="_blank">this protocol</a>.

[GUIDELINES]
<strong>Transformation Protocol Variables</strong><br /><br /><strong>Thawing</strong>:&nbsp;Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0&deg;C will decrease the transformation efficiency.<br /><br /><strong>Incubation of DNA with Cells on Ice</strong>:&nbsp;For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.<br /><br /><strong>Heat Shock</strong>:&nbsp;Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42&deg;C is optimal.<br /><br /><strong>Outgrowth</strong>:&nbsp;Outgrowth at 37&deg;C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.<br /><br /><strong>Plating</strong>:&nbsp;Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.<br /><br /><strong>DNA Contaminants to Avoid</strong><br />
<table width="468">
<tbody>
<tr>
<td><strong>Contaminant</strong></td>
<td><strong>Removal Method</strong></td>
</tr>
<tr>
<td>Detergents</td>
<td>Ethanol precipitate</td>
</tr>
<tr>
<td>Phenol</td>
<td>Extract with chloroform and ethanol precipitate</td>
</tr>
<tr>
<td>Ethanol or Isopropanol</td>
<td>Dry pellet before resuspending</td>
</tr>
<tr>
<td>PEG*</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
<tr>
<td>DNA binding proteins (e.g. Ligase)</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
</tbody>
</table>
<br />* <em>Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 &micro;l of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.</em>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Generating-viral-metagenomes-from-the-coral-holobi-ejgbcjw" target="_blank">Generating viral metagenomes from the coral holobiont</a>.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To ensure reliable quantification of rat lung fibrosis, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition.</p>

[GUIDELINES]
<p>The detection model is working for fixed (under pressure) rat lung paraffin sections cut 2um thick and stained for Masson's trichrome (applicable staining range described in the linked manuscript)</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>Schadenfreude</em> (i.e., the pleasure derived from another’s misfortune) has been widely studied by having participants imagine how they would feel in hypothetical scenarios describing another person’s pain or misfortune. However, research on affective forecasting shows that self-judgments of emotions are inaccurate in hypothetical situations. Here we show a study in which we first presented a hypothetical <em>schadenfreude</em> situation and few months later, due to an exceptional circumstance, the situation turned out to happen in reality. This fortuitous circumstance allowed us to compare people’s imagined emotional reactions with their actual feelings. Results showed that <em>schadenfreude</em> was higher in the real situation than in the hypothetical one. More importantly, participants used different proxies to predict their emotional reaction: while out-group dislike served as a proxy of <em>schadenfreude</em> in both types of scenario, the degree of in-group identification also increased <em>schadenfreude</em> in those who had experienced the real event, arguably a mechanism to promote positive self-evaluation. These results highlight the importance of assessing <em>schadenfreude</em> in the heat of the moment. </p>

[STEPS]
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TITLE: CDO expression into OnePot PURE
AUTHORS: Dana Mozaffari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains the procedure for expressing the catechol degrading enzyme </span><span style = "font-style:italic;">Catechol-2,3-deoxygenase </span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">CDO</span><span>) into a "homemade" OnePot PURE cell-free transcription/translation system.</span></div><div class = "text-block">Once expressed, this enzyme degrades catechol, a colorless substrate, into a yellow colored one, 2-hydroxymuconate semialdyhyde (2-HMS), providing a colorimetric signal that can be easily  implemented in another protocol.</div></div>

[STEPS]
?. Label 3 PCR tubes according to the reactions
?. Add the catechol, CDO and water as needed according to the materials chart.
?. Make a quick spin in the centrifuge to have all the liquid in the bottom.
?. Incubate one hour at 37°C
?. In each tubes add the Energy solution, proteins and ribosomes.
TITLE: Indirect Enzyme Linked Immunosorbent Assay (ELISA) for Detection of Anti-HIV Antibodies in Human Serum
AUTHORS: Norma McFarlane-Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was already used successfully to detect anti-HIV antibody in the serum of women with cervical dysplasia or cervical cancer in Jamaica, West Indies [1].</div><div class = "text-block">Reference</div><div class = "text-block">1. Justiz Vaillant A, Bazuaye PE, McFarlane-Anderson N, Smikle MF et al. Seroprevalence of Anti-HIV Antibodies in Women with Abnormal Pap Smears in Jamaica. British Journal of Medicine & Medical Research 2013, 3(4): 2197-2202.</div></div>

[STEPS]
?. The 96 well polystyrene microplate (U-shaped bottom; Sigma-Aldrich) is coated with 50 ng of a mixture of synthetic peptides (including the fragment 579-601 of the HIV gp41 and fragments 254-274, 308-331 and 421-438 of the HIV gp120) for 4 h at 37ºC.
?. The microplate is blocked with 3% non-fat milk in PBS, 25 µl/well, 1h at room temperature (RT).
?. The microplate is washed 4X with PBS-Tween-20.
?. Duplicates of 25 µl of 1:16 diluted human sera are added.
?. After incubation for 90 min at RT the microplate is washed 4X with PBS-Tween 20.
?. Then, 25 µl of a chimeric commercially-prepared recombinant protein LA-HRP conjugate (Sigma-Aldrich) diluted 1:5000 is added.
?. After incubation for 90 min at RT and rewashing steps 25 µl TMB  is added to each well for 15 min in the dark.
?. The reaction is stopped with 3M H2SO4.
?. The microplate is read in a microplate reader at 450 nm.
?. In the ELISA is included a pooled human sera with high titre of anti-HIV antibodies as positive control, a pooled sera from healthy individuals as negative control and 0.9% normal saline solution was used as the blank.
?. The cut-off point is calculated as mean optical density (XOD) of negative control plus two standard deviation (SD).
TITLE: LC-MS of Native Nanodiscs
AUTHORS: Caroline Brown, Snehasish Ghosh, Rachel McAllister, Mukesh Kumar, Gerard Walker, Eric Sun, Talat Aman, Aniruddha Panda, Shailesh Kumar, Wenxue Li, Jeff Coleman, Yansheng Liu, James E Rothman, Moitrayee Bhattacharyya, Kallol Gupta

[DESCRIPTION]
This protocol provides a step-by-step guide for conducting LC-MS analysis of native nanodiscs extracted from biological membranes.

[STEPS]
SECTION: Liquid Chromatography
1. Resuspend peptides in water + 0.1 formic acid varying amount of water so that 500ng of peptide can be loaded in a reasonable volume.
SECTION: Liquid Chromatography
2. Chromatography is subsequently conducted using home-packed C18 columns (15cm x 75uM ID) for separation of peptides by hydrophobicity.
SECTION: Liquid Chromatography
3. Use a 75 minute separation gradient with a buffer system of water/acetonitrile with 0.1% formic acid beginning at 6% acetonitrile and ending at 100% acetonitrile.
SECTION: Mass Spectrometry
4. Data Dependent Acquisition (DDA) mass spectrometry is conducted using an Orbitrap Eclipse instrument. 
SECTION: Mass Spectrometry
5. MS1 scans a acquired using the Orbitrap detector at a resolution of 120000 and stored in profile mode using a mass range of 350-1400 m/z and and intensity threshold of 1.0e3.
SECTION: Mass Spectrometry
6. Use a cycle time of 1 second for ion isolation and MS2 fragmentation isolating ions with a 1.4 m/z window and fragmented with CID energy at 30% activation. 
SECTION: Mass Spectrometry
7. Product ions are detected at the ion trap set to rapid detection mode with precursors ions being dynamically excluded for 20 seconds after one instance of detection.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>What to look for in a silver stain protocol<br /><br /></strong>Avoid silver-ammine stains. Ammonia is touchy stuff and procedures are lengthy. Glutaraldehyde is unnecessary, unless you've got basic proteins (luteovirus capsids? - no data). Be sure gel is thin (0.75mm). Stain should incorporate a sensitizer (dithionite in my case) and a silver chelator (thiosulfate). The latter prevents silver carbonate  precipitation. Use high quality distilled water in the last stages of development.  Otherwise technical grade chemicals (alcohols, glycine, etc) work fine. I've not  successfully stained tricine gels - even with extensive washing; I presume tricine is a better silver precipitant than glycine.<strong><br /><br />References:<br /><br /></strong>Morrissey, J.H. (1981). Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. <em>Analytical Biochem </em>17 , 307-31.<strong> <br /><br /></strong>Rabilloud, Thierry (2000) Detecting proteins separated by 2-D gel electrophoresis. <em>Analytical Chemistry </em>72 , 48a-55a.<strong> <br /><br />Problem<br /><br /></strong>The protocol is robust. Occasional slow development appears to correlate with old formaldehyde. Doubling the formaldehyde concentration in the developer solves the problem.<strong> <br /> <br /> <br /> <br /> <br /></strong></p>

[STEPS]
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TITLE: Primary cortical neuron isolation and culture
AUTHORS: Shiyi Wang

[DESCRIPTION]
Primary cortical neuron isolation and culture

[STEPS]
1. **Dissection and Digestion** - Micro-dissect cortices from P1 rat pups of both sexes (Sprague Dawley, Charles River Laboratories, SD-001). - Digest cortices in papain (~7.5 units/ml) at 33°C for 45 minutes.
2. **Cell Preparation** - Triturate the digested tissue in low and high ovomucoid solutions. - Resuspend cells in panning buffer (DPBS (GIBCO 14287) supplemented with BSA and insulin). - Pass cells through a 20 μm mesh filter (Elko Filtering 03-20/14).
3. **Negative Panning** - Incubate filtered cells on negative panning dishes coated with: - Bandeiraea Simplicifolia Lectin 1 (x2) - Goat anti-mouse IgG+IgM (H+L) (Jackson ImmunoResearch 115-005-044) - Goat anti-rat IgG+IgM (H+L) (Jackson ImmunoResearch 112-005-044) antibodies.
4. **Positive Panning** - Incubate cells on positive panning dishes coated with mouse anti-L1 (ASCS4, Developmental Studies Hybridoma Bank, Univ. Iowa) to bind cortical neurons. - Collect adherent neurons by forceful pipetting with a P1000 pipette.
5. **Neuron Isolation and Plating** - Pellet isolated neurons (11 minutes at 200 g). - Resuspend neurons in serum-free neuron growth media (NGM): - Neurobasal, B27 supplement, 2 mM L-Glutamine, 100 U/ml Pen/Strep, 1 mM sodium pyruvate, 4.2 μg/ml Forskolin, 50 ng/mL BDNF, and 10 ng/mL CNTF. - Plate 70,000 neurons onto 12 mm glass coverslips coated with 10 μg/ml poly-D-lysine (PDL, Sigma P6407) and 2 μg/ml laminin. - Incubate at 37°C in 10% CO2.
6. **Media Changes and Maintenance** - On day in-vitro (DIV) 2: - Replace half of the media with NGM Plus (Neurobasal Plus, B27 Plus, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 4.2 μg/ml Forskolin, 50 ng/ml BDNF, and 10 ng/ml CNTF). - Add AraC (10 μM) to stop the growth of proliferating contaminating cells. - On DIV 3: - Replace all the media with NGM Plus. - On DIV 6 and DIV 9: - Replace half of the media with NGM Plus.
7. **Lentivirus Infection Protocol** - On DIV 2: - Add 100 μl of supernatant containing lentivirus plus polybrene (1 μg/ml) to the AraC NGM mixture. - On DIV 3: - Completely wash out the lentivirus and replace with NGM Plus containing 100 ng/ml BDNF.