eamag/protocols-with-steps huggingface dataset:
TITLE: TUNEL
AUTHORS: 张 雪

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

[STEPS]
?. [Day1]
复水：75%MetOH+25%PBT 1ml shake for 5min
?. [Day1]
50%MetOH+50%PBT 5min
?. [Day1]
25%MetOH+75%PBT 5min
?. [Day1]
100%PBT 5min 1/4
融化PK
?. [Day1]
100%PBT 5min 2/4
?. [Day1]
100%PBT 5min 3/4
?. [Day1]
100%PBT 5min 4/4
?. [Day1]
透化：Proteinase K: PBT = 1 : 2000 (终浓度5µg/ml），5min
?. [Day1]
再固定：4%PFA 20min
加PFA时，加完一管应立即放在摇床上，否则容易在底部凝结成团
?. [Day1]
1×PBT 5min 1/3
?. [Day1]
1×PBT 5min 2/3
?. [Day1]
1×PBT 5min 3/3
?. [Day2]
回收一抗，保存在4°C冰箱
?. [Day2]
PT 20min 1/8
?. [Day2]
PT 20min 2/8
?. [Day2]
PT 20min 3/8
?. [Day2]
PT 20min 4/8
?. [Day2]
PT 20min 5/8
?. [Day2]
PT 20min 6/8
?. [Day2]
PT 20min 7/8
?. [Day2]
PT 20min 8/8（此后直接孵育二抗，不用加PBTN blocking）
?. [Day2]
＋二抗（in PBTN，1:2000，200µl，4°C过夜）： 例donkey−goat 633，donkey-rabbit 488，donkey-rabbit 633 二抗与对应的所有一抗的来源都不能相同
200 µl
4 °C
?. [Day3]
吸去二抗，不回收
?. [Day3]
PT 20min 1/8
?. [Day3]
PT 20min 2/8
?. [Day3]
PT 20min 3/8
?. [Day3]
PT 20min 4/8
?. [Day3]
PT 20min 5/8
?. [Day3]
PT 20min 6/8
?. [Day3]
PT 20min 7/8
?. [Day3]
PT 20min 8/8
?. [Day3]
直接拍照或1×PT在4°C摇床洗过夜，后者可以去除很多杂背景
?. [Day1]
100%PBT 5min 1/2
?. [Day1]
100%PBT 5min 2/2
?. [Day1]
渗透：丙酮:乙醇（1:2）,  -20°C，30min
-20 °C
?. [Day1]
预杂交：+45µL底物（equilibration buffer，Rhoche，红盖），37°C，2～3h，此后避光
37 °C
此后所有操作避光
?. [Day1]
杂交：+5µL酶（Rhoche，蓝盖），37°C，1～1.5h
37 °C
?. [Day1]
2×SSCT，37°C，20min 1/2
37 °C
?. [Day1]
2×SSCT，37°C，20min 2/2
37 °C
?. [Day1]
2×SSCT，室温摇床，20min
0 Room temperature
?. [Day1]
1×PBT 5min 1/3
?. [Day1]
1×PBT 5min 2/3
?. [Day1]
1×PBT 5min 3/3
?. [Day1]
+blocking PBTN 500µl，4°C摇床，2h
4 °C
500 µl
?. [Day1]
+一抗（抗体:PBTN=1:1000，100µl，4°C摇床过夜） Dendra2：Rabbit-anti-Dendra2／ Kaeda：Rabbit-anti-kaeda ／ Mcherry：Mouse-anti-mcherry ／ CFP/GFP：Ab6658（goat），A1122（rabbit），Sc9996（mouse ）／ DsRed：Mouse-anti-DsRed 若实验对象带有两种及以上荧光，注意两种抗体应选择不同的来源
100 µl
4 °C
TITLE: Publication and Data Sharing Policy (Part 13 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 13 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: Indoor active search for adult Aedes sp. and Culex sp. mosquitoes
AUTHORS: Erika  Santamaria, Paola  Muñoz, Catalina Marceló

[DESCRIPTION]
This protocol details the indoor active search carried out in the research project: "Spatial stratification of dengue based on the identification of risk factors: a pilot trial in Cauca, Colombia"

Previous studies have reported that, indoors, adult Aedes aegypti mosquitoes rest on surfaces such as walls and furniture and generally at a height of less than 1.5 m (Dzul-Manzanilla et al., 2016 & Chadee, 2013). The use of electromechanical aspirators such as the Prokopack aspirator has also been reported (Vazquez-Prokopec et al., 2009) which allows for a relatively easy collection of resting mosquitoes.


STEPS: 

1) Mosquito collection: Adult mosquitoes will be captured with the Prokopack aspirator at potential resting places inside the dwellings (walls, curtains, behind furniture, behind pictures, behind TV sets, under beds), between 480 min AM and 300 min PM. Collection shall be carried out for 10-15 minutes inside the household (bedrooms and living rooms).

As information of interest for the sampling, most of the houses in the urban areas of Piamonte, Patía and Miranda, Cauca are one-storey houses, but there are also houses with two or even three floors. The walls are generally made of brick plus plaster (a mixture to cover a wall), the roof is made of clay tiles and the floors are made of cement; however, there are more precarious dwellings built with wood walls and roofs (Alcaldía de Patía, 2016).

2) In each household, an e-form designed using ArcGIS 10.8 will be completed with data on the location of the household, predominant construction materials and the results of entomological sampling for adults. 


3) Storage of mosquitoes: Collected mosquitoes can be immobilised with chloroform vapour (by placing them Prokopack collection bottle in a plastic bag with cotton wool soaked in chloroform). 

4) Mosquitoes should be stored carefully and kept cold at 4 °C until identification.  The vials shall be labelled with the main data such as locality code, house code and date of collection.

4) Pooling: Collected females identified as Ae. aegypti will be pooled in pools of a maximum of 10 adult female mosquitoes per 0.2 mL vial with buffer  RNA Later® (Dos-reis et al., 2019). The cold chain will be maintained until reaching the INS Entomology Laboratory for subsequent RNA extraction (Pérez-Castro et al.¸2016 & Pham et al., 2017).


[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a method for identifying bacteriophage/virus taxonomy without a virome dataset using UniProt reference database. Based on the methods found in the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplementary information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software:</strong><br />
<ul>
<li>NCBI's BLAST+ v2.2.0</li>
<li>UniProt Database</li>
<li>Bowtie2-2.1.0</li>
<li>MEGAN-5.5.3</li>
</ul>
<strong>Relevant Files</strong><br /><br />Output:<br />
<ul>
<li>Bray-curtis_virome_analysis/contig_otu_table_transposed_formatted.txt</li>
<li>Phage_Taxonomy/order_rel_abund.tsv</li>
<li>Phage_Taxonomy/genus_rel_abund.tsv</li>
<li>Phage_Taxonomy/species_rel_abund.tsv</li>
</ul>
Perl Scripts:<br />
<ul>
<li>remove_block_fasta_format.pl</li>
<li>filter_fasta_file.pl</li>
<li>get_format_order.pl, get_format_family.pl, get_format_subfamily.pl, get_format_genus.pl</li>
<li>contig_id_by_orfs.pl</li>
<li>calculate_abundance_from_sam.pl</li>
</ul>
Python script:<br />
<ul>
<li>transpose_tab_delim.py</li>
</ul>
R scripts: <br />
<ul>
<li><a href="https://www.protocols.io/view/Script-R1-Virome-Contig-and-Sequencing-Statistics-eh5bb86?step=undefined" target="_blank">R1</a> and <a href="https://www.protocols.io/view/Script-R4-Virome-Taxonomy-eiabcae" target="_blank">R4</a></li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: IHC-Amplified Fluorescent Frozen Sections
AUTHORS: Elizabeth Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures and amplify specific antibody signals.</div></div>

[STEPS]
?. Day 1: Using the PAP Pen, carefully draw a water barrier circle around the tissue sections on the slide – allow this circle to dry for several seconds or up to approx. one minute
?. Rinse slides with PBS (pH 7.3-7.4): 4 x 5 min each
?. Rinse slides with 0.5% BSA + 0.4% Triton X-100 in PBS):  1 x 10 min
?. Remove slides one at a time and using a clean Kimwipe, carefully wipe around the tissue sections to dry the slide
?. Place the slides into a black, covered slide incubation box/humidity box
?. Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
?. Pour off the blocking buffer
?. Replace with primary antibody solution (antibody of choice diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Incubate tissue with primary antibody overnight in incubation box
?. Day 2: Rinse slides with PBS:  4 x 5 min
?. Rinse slides with 0.5% BSA + 0.4% Triton X-100 + PBS:  1 x 10 min
?. Place the slides into a black, covered slide incubation box/humidity box
?. Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
?. Prepare fluorescent secondary antibody (secondary antibody should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Cover the tissue with the secondary antibody solution and incubate for 2 hrs. at RT in the incubation box. *From this point on, use low light and/or cover tissues.*
?. Rinse slides with PBS:  4 x 5 min
?. Remove excess PBS with a Kimwipe
?. Carefully add a drop of mounting medium to the center of the tissue and apply cover glass
?. Seal cover glass with clear nail polish. For thicker tissue, add a weight before sealing
?. Prepare fluorescent Streptavidin secondary antibody (should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
?. Cover the tissue with the fluorescent Streptavidin antibody solution and incubate for 2 hrs. at RT in the incubation box.
?. Rinse slides with PBS:4 x 5 min
?. Remove excess PBS with a Kimwipe
TITLE: Expression and purification of PI3KC3-C1 complex
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
Expression and purification of PI3KC3-C1 complex

[STEPS]
SECTION: Expression
1. Transfect HEK GNTi cells at concentration of 2 × 10^6 cells/ml
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Expression
3. In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
4. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C

SECTION: Expression
5. Add mixture to cells. Let cells grow for 2880 min

SECTION: Expression
6. Harvest Cells 500 rpm, 4 °C, 10 min

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at-80 °C until purification. 

SECTION: Purification
8. Pellets were homogenized 20 times by Pyrex douncer (Corning) in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 10% Glycerol) with 25mM TCEP/proteinase inhibitors (Thermo Scientific), and then add 10% Triton X-100 stock to final 1% concentration.  
SECTION: Purification
10. Clarify lysate for 17000 rpm for 40 min at 4 °C.  




SECTION: Purification
11. Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin
SECTION: Purification
12. Rock supernatant with equilibrated Strep-Tactin Sepharose resin for 60 min at 4 °C
SECTION: Purification
13. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 25 mM TCEP, 10% Glycerol)
SECTION: Purification
14. Elute with lysis buffer plus 4 mM desthiobiotin for STREP resin
SECTION: Purification
15. Concentrate elution and inject onto pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva) 
 (25 mM HEPES pH 7.5, 300 mM NaCl, 1 mM MgCl2, 1 mM TCEP)
SECTION: Purification
16. Pool peak fractions, concentrate, snap freeze, and store at -80 °C 

SECTION: Purification
9. Rocking at 4 °C for60 min
TITLE: Qubit DNA Quantification (Assay)
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
Achieve accurate and precise quantification of dsDNA with Qubit dsDNA HS (High Sensitivity) and Qubit dsDNA BR (Broad Range) Assay Kits.  These dsDNA quantification kits enable quick and selective detection of low and high abundance DNA samples, and can distinguish dsDNA from ssDNA, RNA, protein, and free nucleotides. Contaminants, such as salts, solvents, or detergents are well-tolerated.

[BEFORE_START]
Qubit dsDNA HS and BR Assay kits can be used with any Qubit Fluorometer.

Use with thin-wall, clear, 0.5-mL PCR tubes (Cat. No. Q32856) for the Qubit 4 Fluorometer and 8 x 200-µL tube strips (Cat. No. Q33252) for the Qubit Flex Fluorometer

[GUIDELINES]
Qubit dsDNA HS Assay Kit

The Qubit dsDNA HS (High Sensitivity) Assay Kit, when used with the Qubit Fluorometer, provides an accurate and selective method for the quantitation of sensitive DNA samples.  Depending on sample volume, the assay kit is designed to be accurate for initial DNA sample concentrations of 0.005 to 120 ng/µL, providing a detection range of 0.1−120 ng.

Qubit dsDNA BR Assay Kit

The Qubit dsDNA BR (Broad-Range) Assay Kit, when used with the Qubit Fluorometer, provides an accurate and selective method for the quantitation of DNA samples.  Depending on sample volume, the assay kit is designed to be accurate for initial DNA sample concentrations of 0.2 to 2,000 ng/µL, providing a detection range of 4−2,000 ng.

[STEPS]
SECTION: Preparing Working Solution + Samples
1. Clean the workstation with DNA Away and Ethanol.
SECTION: Preparing Working Solution + Samples
2. Remove the PCR plates from the -20 °Cfreezer to thaw.

SECTION: Preparing Working Solution + Samples
3. Prepare two assay tubes for the upper and lower bound standards, and also one tube for each sample to be tested or quantified for DNA concentration.
SECTION: Preparing Working Solution + Samples
3.1. Prepare the working solution (can be calculated using the calculator on the Qubit instrument):

Prepare the assay tubes as follows: 

Standards: 190 µL working solution + 10 µL standard from kit

Samples: 198 µL working solution + 2 µL sample

SECTION: Preparing Working Solution + Samples
4. Vortex all tubes for 3 s.

SECTION: Preparing Working Solution + Samples
5. Incubate the tubes for 2 min at Room temperature.

SECTION: Running Qubit
6. On the Qubit 2.0 Fluorometer, select dsDNA High Sensitivity or dsDNA Broad Sensitivity.
SECTION: Running Qubit
7. Run two standards as indicated by the program.
SECTION: Running Qubit
8. Insert the tubes into the instrument and record the amplicon values.
SECTION: Running Qubit
9. Once finished, throw all assay tubes into the Qubit disposal bin and clean the workstation.
SECTION: Running Qubit
10. Return all PCR plates to the -20 °C freezer for storage.
TITLE: Fluorescently labeled polyamine uptake (via Flow Cytometry)
AUTHORS: Marine Houdou, Peter Vangheluwe

[DESCRIPTION]
Assess polyamine uptake capacities of a specific cell line after incubation with fluorescently labeled polyamines and mean fluorescence intensitiy acquisition via flow cytometry. 

[BEFORE_START]
Prepare appropriate dilution of fluorescently labeled polyamine in cell culture medium.

Prepare Flow Cytometry (FC) buffer made of 1% Albumin Fraction V in DPBS (-/-).

Prepare Eppendorf tubes and FC tubes by labeling them and keeping them on ice. 

[GUIDELINES]
Make sure to stay protect from the light when manipulating fluorescent materials.

[STEPS]
2. The day of the experiment, remove cell culture medium and add fluorescently labeled polyamines to the cells in a final volume of 500 µL. 
 

 


3. Incubate the desired time at 37°C, 5% CO2, protected from the light.
4.1. Discard medium.
4.2. Wash with 500 µL Versene or DPBS (-/-).

4.5. Add 500 µL 1%-Albumin Fraction V in DPBS (-/-), collect the cells and transfer in Eppendorf tubes on ice. 
4.6. Centrifuge for 5 minat 400 g and 4°C.

4.7. Discard supernatants and resuspend cell pellets in 250-500 µL 1%-Albumin Fraction V in DPBS (-/-), depending on the size of the cell pellet.

5. Keep on ice until acquisition at the flow cytometer. Record 10,000 live events per sample.
4.4. Add 200 µL 0.25% Trypsin or TrypLE. Incubate 5 min protect from the light.


1. Seed cells in 12 well-plate that they reach 70%-80% confluency the day of the experiment.
 

4. Harvest cells and prepare samples for Flow Cytometry (FC).
4.3. Discard Versene or DPBS (-/-).
4.8. Filter cell suspension through Nylon filter into FC tubes.
TITLE: AN OBSERVATIONAL STUDY ON THE EFFICACY OF DAPAGLIFLOZIN IN HEART FAILURE WITH REDUCED EJECTION FRACTION IN TERTIARY CARE HOSPITAL IN CENTRAL INDIA
AUTHORS: Meghna Bordoloi, Shilpa Bawankule, Sourya Acharya, Sunil Kumar

[DESCRIPTION]
Heart failure with reduced ejection fraction is a prevalent and debilitating condition associated with significant mortality and morbidity. Dapagliflozin, an SGLT2 inhibitor shows promising results in reducing cardiovascular events and improving clinical outcomes. However, Real world evidence of it’s efficacy and safety is limited. This observational study aims to study the effects of dapagliflozin in patients with heart failure with reduced ejection fraction. It expects to see reduced number of recurrent hospitalisation, cardiovascular deaths, improved NYHA class, and improvement in cardiometabolic parameters as well study the adverse effects that follows. 

[STEPS]
1.
TITLE: Qiagen QIAmp PowerFecal Pro extraction kit
AUTHORS: Michael Dan Siemon, Dilan Joseph, Jessica Pardy, Richard Gibson, christopher.degroot

[DESCRIPTION]
This protocol is used by Western University to process wastewater samples for wastewater-based epidemiology. The protocol is modified from the protocol described in the RNeasy PowerFecal Pro Kit Handbook provided by Qiagen with the PowerFecal Pro kit. 

[STEPS]
SECTION: Sample Preparation
1. Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 4500 x g for 120 min. Decant supernatant, assume 280 µl pellet.
SECTION: Sample Preparation
4. As a substitute for vortexing described in the kit protocol, bead-beating is used. Bead-beating is conducted 4x for 30s at 4 m/s.
SECTION: Sample Preparation
3. Add 650 μl of Solution CD1 to the wastewater pellet and transfer solution to the PowerBead Pro Tube.
SECTION: Sample Preparation
5. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.
SECTION: Sample Preparation
6. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube. Add 200 μl of Solution CD2 and vortex for 5 s.
SECTION: Sample Preparation
7. Centrifuge at 15,000 x g for 1 min. Avoiding the pellet, transfer up to 650 μl of supernatant to a clean 2 ml Microcentrifuge Tube.
SECTION: Sample Preparation
8. Add 650 μl of Solution EA. Vortex briefly.
SECTION: Sample Preparation
9. Load 650 μl supernatant-EA mix into an MB RNA Spin Column and centrifuge at 15,000
x g for 1 min. Discard the flow-through and repeat until all of the solution has been passed through the Spin Column.
SECTION: Sample Preparation
10. Add 650 μl Solution EA to the Spin Column and centrifuge at 15,000 x g for 1 min. 
SECTION: Sample Preparation
11. Mix 45 μl DNase Digestion Solution and 5 μl DNase I stock enzyme. Place the MB RNA Spin Column into a clean 2 ml Collection Tube and add the DNase Digestion Solution to the center of the filter. Incubate at room temperature for 15 minutes.
SECTION: Sample Preparation
12. Add 650 μl Solution EA to the Spin Column and centrifuge at 15,000 x g for 1 min. 
SECTION: Sample Preparation
13. Discard the flow-through. Add 500 μl Solution C5. Centrifuge at 15,000 x g for 1 min.
SECTION: Sample Preparation
14. Replace the collection tube and dry the Spin Column by centrifuging at 20,000 x g for 1 min.
SECTION: Sample Preparation
15. Elute the RNA by placing the spin column in a 1.5 ml collection tube. Add 100 μl of RNase-free water and centrifuge at 15,000 x g for 1 min.
SECTION: Sample Preparation
2. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add 100 μl  of phenol−chloroform−isoamyl alcohol to the PowerBead Pro Tube.
TITLE: IPA assembly for PacBio Hifi reads
AUTHORS: a.kharabianmasouleh , priyanka.sharma , Othman  Aldossary, Bader  Alsubaie, Onkar  Nath, Valentine  Murigneux, Neena  Mitter, Gabriel  Rodrigues Alves Margarido, Bruce  Topp, imssallem , Agnelo  Furtado, Robert Henry

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is regarding the assembly of plant genomes using HiFi data (PacBio Sequel II) with Improved Phase Assembly (IPA) tool.</div></div>

[STEPS]
?. Steps:1) ccs.bam files were generated by PacBio Sequel II (Hifi) platform for all the plant species, used to assemble the genome using IPA tool.2) The IPA tool was installed using the conda environment, following the link:https://github.com/PacificBiosciences/pbipa
?.
TITLE: PNGase F Protocol (Denaturing Conditions)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
PNGase F is the most effective enzymatic method for removing almost allN-linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides. 

This is a generic PNGase F protocol for denaturing reaction conditions. It is appropriate for both P0704 and P0708. 

  


[BEFORE_START]
Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:

[GUIDELINES]
If using P0704/P0708, we recommend limiting PNGase F to 1/10 (or less) of the total reaction volume to keep the final glycerol concentration equal to (or less than) 5%.
For unit conversion between different suppliers, please reference the Glycobiology Unit Conversion Chart page.

Biology Unit Conversion Chart
Reagent companies differ in how a unit of enzyme is defined. This chart can be used to help determine how a unit of enzyme from one company compares to a unit of enzyme from NEB. All enzymes were assayed using NEB's assay protocols as a means of normalization (NEB Assay).
EnzymeCompanySelling Conc. (U/ml)Units /Vialµl/VialNEB Assay (U/ml)NEB Assay Units /Vialµl Conversion (1 NEB µl = x Company µls)PNGase FNEB (NEB #P0704/P0705)500,00015,00030500,00015,0001Prozyme (GKE-5006A)2.50.140150,0006,0003.3Prozyme (GKE-5020B, Ultra)100.440500,00020,0001QA Bio (E-PNG01)50.360200,00012,0002.5Sigma (P7367)500505090,0004,5005.5


[STEPS]
SECTION: Denaturing Reaction Conditions:
1. Combine 1 µg - 20 µg, 1 µL and H2O (if necessary) to make a 10 µL total reaction volume.
SECTION: Denaturing Reaction Conditions:
2. Denature glycoprotein by heating reaction at 100 °C for 10 min.
SECTION: Denaturing Reaction Conditions:
3. Chill denatured glycoprotein on ice and centrifuge 10 s.
SECTION: Denaturing Reaction Conditions:
4. Make a total reaction volume of 20 µL by adding: 2 µL, 2 µL and 6 µL. 
 

SECTION: Denaturing Reaction Conditions:
5. Add 1 µL and mix gently.
SECTION: Denaturing Reaction Conditions:
6. Incubate reaction at 37 °C for 60 min.
SECTION: Denaturing Reaction Conditions:
7. Analyze by method of choice.