eamag/protocols-with-steps huggingface dataset:
TITLE: Inhibitor removal from DNA extracts
AUTHORS: Dominik Buchner, Marie Borowski

[DESCRIPTION]
This protocol describes how to remove inihibitory substances such as humic substances from DNA that has already been extracted. The protocol is formulated for an initial input of 100 µL of extracted DNA however it can be adjusted to any other volume as well with minor changes. A lot of the buffers can be found in the following patent https://patents.google.com/patent/US7459548B2/en


[BEFORE_START]
Make sure all buffers are prepared before starting.

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.

[STEPS]
SECTION: Inhibitor removal from DNA extracts
1. Prepare 100 µL of sample in 2 mL tubes.

 

 

SECTION: Inhibitor removal from DNA extracts
2. Add 345 µL and 60 µL .
Vortex shortly.
SECTION: Inhibitor removal from DNA extracts
3. 10000 x g, 20 °C . Transfer all of the supernatant to a new tube.

 

SECTION: Inhibitor removal from DNA extracts
4. Add125 µL , vortex shortly and incubate at 4 °C for 5 min .

 

SECTION: Inhibitor removal from DNA extracts
7. 10000 x g, 1 min, 20 °C . Transfer600 µL of the supernatant to a new tube.

SECTION: Inhibitor removal from DNA extracts
8. Add1200 µL . Vortex to mix.

SECTION: Inhibitor removal from DNA extracts
9. Load 650 µL of the mixture to a mini spin column (e.g. Epoch Life Science).

 


SECTION: Inhibitor removal from DNA extracts
10. 10000 x g, 30 s, 20 °C . Discard the flow-through. Repeat two times to bind the complete sample volume. 

SECTION: Inhibitor removal from DNA extracts
11. Add 500 µL . 10000 x g, 30 s, 20 °C to wash the column. Discard the flow-through.

SECTION: Inhibitor removal from DNA extracts
12. 10000 x g, 1 min, 20 °C to dry the column. Transfer the spin column to a clean 1.5mL microcentrifuge tube. 

SECTION: Inhibitor removal from DNA extracts
13. Add50 µL . Incubate for 3 min atRoom temperature .

SECTION: Inhibitor removal from DNA extracts
14. 10000 x g, 1 min, 20 °C to eluate the DNA. DNA eluate should be completely colorless and ready to go for downstrom analysis.

 


SECTION: Inhibitor removal from DNA extracts
5. 10000 x g, 1 min, 20 °C . Transfer the supernatant to a new tube.

SECTION: Inhibitor removal from DNA extracts
6. Add 100 µL . A precipitate may form. Vortex shortly, incubate at4 °C for5 min
TITLE: Expression and purification ATG13, ATG101 and FOLDON-ATG9A Proteins from HEK293GnTi Cells 
AUTHORS: Adam  Yokom

[DESCRIPTION]
Expression and purification from HEK cells of ATG13, ATG101 and FOLDON-ATG9A proteins

[STEPS]
SECTION: Expression
1. Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
   
SECTION: Purification
8. Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA) 
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Purification
9. Clarify lysate for 17000 rpm, 30 min, 4 °C

SECTION: Purification
10. Rock supernatant with equilibrated resin for 60 min at 4 °C
    Use glutathione resin for GST tagged proteins
    Use amylose resin for MBP tagged proteins
    Use Strep-Tactin Sepharose resin for Strep tagged proteins
SECTION: Purification
11. Let supernatant Flow thru gravity column
SECTION: Purification
12. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
SECTION: Purification
14. Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA)
SECTION: Purification
13. Elute with lysis buffer plus elution additive 
    25 mM glutathione for GST resin
    40 mM Maltose for amylose resin
    4 mM desthiobiotin for STREP resin
SECTION: Purification
15. Pool peak fractions, concentrate, snap freeze, and store at -80C 
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, 10 min, 4 °C 

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at -80C until purification.
TITLE: Standard Operating Procedure for Solid Phase Adsorption Toxin Testing (SPATT) Assemblage and Extraction of HAB Toxins v2
AUTHORS: Kendra Negrey, Meredith Howard, Jayme Smith, Raphael Kudela, David Caron

[DESCRIPTION]
SPATT has been developed and tested for a variety of resins (see review by Kudela, 2017). These Standard Operating Procedures (SOP) outline the use of the resin DIAON HP20 since it has been demonstrated to quantify microcystins, anatoxin-a, saxitoxin, domoic acid, and okadaic acid in fresh, brackish, and marine waters, all from the same SPATT sampling device (Lane et al. 2010; Miller et al. 2010, Kudela 2011, Gibble and Kudela, 2014, Howard et al., 2017, Kudela, 2017, Peacock et al., 2018). Cylindrospermopsin and nodularin have also been detected using SPATT with DIAON HP20 resin in California, however, the use of DIAON HP20 for these toxins has not been well characterized in the laboratory. This methodology is ready for wider adoption by the research, monitoring, and management communities interested in detecting and tracking the dynamics of freshwater and marine toxins. The SOP described herein for the assemblage and construction of SPATT bags can be used for deployment in freshwater, brackish and marine environments and the extraction process described below is for the analysis of both marine toxins and cyanotoxins.

[STEPS]
TITLE: EXPRESSION AND PURIFICATION OF HUMAN NEMO (GST-GFP-NEMO)
AUTHORS: Elisabeth Holzer

[DESCRIPTION]
This protocol describes how to express and purify human NEMO (IKK-γ) tagged N-terminally with GST and EGFP. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a GST batch purification and gel filtration (SEC). 

[BEFORE_START]
Lysis Buffer:
50 mM HEPES pH 7.5
300 mM NaCl
2 mM MgCl2
Freshly added: 2 mM β-Mercaptoethanol, Roche Protease Inhibitor (Merck, #5056489001), and DNAse I (Sigma, #DN25-1G)

Wash Buffer 1:
50 mM HEPES pH 7.5
300 mM NaCl
Freshly added: 1 mM DTT

Wash Buffer 2:
50 mM HEPES pH 7.5
700 mM NaCl
Freshly added: 1 mM DTT

SEC Buffer:
25 mM HEPES pH 7.5
150 mM NaCl
Freshly added: 1 mM DTT



[STEPS]
SECTION: Expression
1. Grow E. coli Rosetta (DE3) pLysS cells in 2 L LB medium at 37 °C until achieving an OD600 nm of 0.4. 

SECTION: Expression
2. Reduce temperature to 18 °C and continue growth to OD600 nm of 0.8. 

SECTION: Expression
3. Induce protein expression with 250 micromolar (µM) IPTG and continue growth for ~960 min at 18 °C 

SECTION: Expression
4. Centrifuge the cells at 3000 rcf (4 °C ,15 min )

SECTION: Expression
5. Aspirate media and resuspend cell pellet in ~ 10 ml of 1X PBS, centrifuge the cells at 3000 rcf (4 °C ,15 min ) and take off the supernatant. 
SECTION: Expression
6. Flash freeze the pellet in liquid nitrogen and store at -80 °C until use.

6. 
SECTION: Purification
7. Thaw pellet and resuspend in Lysis Buffer with freshly added 2 millimolar (mM) β-Mercaptoethanol, Roche Protease Inhibitor and DNAse I

SECTION: Purification
8. Sonicate sample 30 s at 65% power for 5 cycles (Bandelin Sonopuls)

SECTION: Purification
9. Centrifuge at 48000 rcf at 4 °C for 45 min

SECTION: Purification
8.1. Repeat sonication for a total of 3X 
SECTION: Purification
11. 
Incubate the sample on equilibrated beads on a tube roller at 4 °C for 240 min 

SECTION: Purification
9.1. During this step, equilibrate 3 mL Glutathione Sepharose 4B beads by washing the slurry with water and Wash Buffer 1 (~ 6 ml).
SECTION: Purification
10. Filter supernatant after centrifugation through  a   syringe filter 
SECTION: Purification
12. Wash beads 5X with Wash Buffer 1
SECTION: Purification
13. Wash beads 1X with Wash Buffer 2

 

SECTION: Purification
14. Wash beads 2X with Wash Buffer 1
 

SECTION: Purification
15. Cleave the resulting sample to produce GFP-NEMO by incubating with thrombin 240 min at 4 °C

SECTION: Purification
16. Centrifuge beads at 3000 rcf for 3 min at 4 °C

SECTION: Purification
17. Collect supernatant and filter through   syringe filter

SECTION: Purification
18. Concentrate product to a final volume of 500 µL using a 10 kDa MWCO concentrator

SECTION: Purification
19. The concentrated and filtered protein can be applied onto a Superose 6 increase column (10/300 Cytiva) pre-equillibrated with SEC buffer
 

SECTION: Purification
20. Fractions containing the purified proteins are pooled, concentrated, frozen in liquid nitrogen and stored at - 80°C
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Prepare appropriate stock-solutions of peptide according to the concentration added in the first well of the microtiter plate. </p>

[GUIDELINES]
<p>Be careful not to contaminate neighbouring wells/rows with the peptide added. If the peptides are highly potent it is a good idea to separate different peptides added (or at different starting-concentrations) by an empty row in-between.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunohistochemistry of tissue sections from formalin-fixed paraffin embedded (FFPE) samples
AUTHORS: Terri Li, Can Gong

[DESCRIPTION]
H&E staining and immunohistochemistry of tissue sections from formalin-fixed paraffin embedded (FFPE) murine tissue samples were performed by the University of Chicago Human Tissue Resource Center core facility.

[STEPS]
1. Biotinylated anti-mouse Tn IgM antibody (clone 5F4) and WE scFv staining
1.1. Tissue sections were deparaffinized and rehydrated through xylenes and serial dilutions of ethanol to deionized water.
1.2. Tissue sections were incubated in antigen retrieval buffer (DAKO S2367) and heated in a steamer at over 97'C for 20 minutes.
1.3. Biotinylated anti-Tn antibody (1:20) or biotinylated WE scFv (1:200) were applied to tissue sections for 1 hour at room temperature.
1.4. After washing, the biotinylated reagent was detected with the Elite kit (Vector Laboratories PK-6100) and DAB (DAKO K3468) system.
2. Anti-CD3 (Abcam ab135372 clone SP162), anti-F4/80 (BioRad MCA497GA clone A3-1), anti-Ly6G (BioLegend 127602 clone 1A8) and anti-cleaved Caspase 3 (Cell Signaling Technology 9661)  staining
2.1. The slide was stained using Leica Bond RX automated stainer.
2.2. After deparaffinization and rehydration, tissue section was heat treated for 20 minutes with antigen retrieval solution (Leica Biosystems AR9961).
2.3. Respective antibody (1:100) was applied on tissue sections for 60 minutes at room temperature and the antigen-antibody binding was detected with Bond Polymer Refine Detection (Leica Biosystems DS9800) without post-primary reagent.
3. Tissue sections were stained with hematoxylin for counterstaining and were covered with cover glasses.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Primers:</strong><br /><br />926F:     AAA CTY AAA KGA ATT GAC GG<br />1492R:   TAC GGY TAC CTT GTT ACG ACT T<br />OR<br />GM3:     AGA GTT TGA TCM TGG C        These primers are for almost full-length 16s (8F/1492R)<br />GM4:     TAC CTT GTT ACG ACT T<br /><br /><strong>Reaction Mix for 926F/1492R:</strong><br /><br />
<table>
<tbody>
<tr>
<td>Water</td>
<td>16.25μl</td>
</tr>
<tr>
<td>10x LA Buffer</td>
<td>2.5μl</td>
</tr>
<tr>
<td>dNTP mix, 2.5mM each</td>
<td>4.0μl</td>
</tr>
<tr>
<td>Primer 926F, 10μM</td>
<td>0.5μl</td>
</tr>
<tr>
<td>Primer 1492R, 10μM</td>
<td>0.5μl</td>
</tr>
<tr>
<td>LA Takara HS 2.5U/μl</td>
<td>0.25μl</td>
</tr>
<tr>
<td>DNA</td>
<td>1.0μl</td>
</tr>
<tr>
<td>Total</td>
<td>25μl</td>
</tr>
</tbody>
</table>
<br /><strong><strong>Cycling with 926F/1492R:<br /><br /></strong></strong>
<table>
<tbody>
<tr>
<td>95°C</td>
<td>3min</td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>95°C</td>
<td>30sec</td>
<td rowspan="3"><br />25 cycles<br /><br /></td>
</tr>
<tr>
<td>55°C</td>
<td>45sec</td>
</tr>
<tr>
<td>72°C</td>
<td>90sec</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>72°C</td>
<td>10min</td>
<td> </td>
</tr>
<tr>
<td>15°C</td>
<td>Hold</td>
<td> </td>
</tr>
</tbody>
</table>
<br /><strong>Reaction Mix for GM3/GM4:</strong><br /><br />
<table>
<tbody>
<tr>
<td>Water</td>
<td>17.875μl</td>
</tr>
<tr>
<td>10x LA Buffer</td>
<td>2.5μl</td>
</tr>
<tr>
<td>dNTP mix, 2.5mM each</td>
<td>2.0μl</td>
</tr>
<tr>
<td>Primer GM3, 100μM</td>
<td>0.25μl</td>
</tr>
<tr>
<td>Primer GM4, 100μM</td>
<td>0.25μl</td>
</tr>
<tr>
<td>LA Takara HS 2.5U/μl</td>
<td>0.125μl</td>
</tr>
<tr>
<td>DNA</td>
<td>2.0μl</td>
</tr>
<tr>
<td>Total</td>
<td>25μl</td>
</tr>
</tbody>
</table>
<br /><strong>Cycling with GM3/GM4:</strong><br /><br />
<table>
<tbody>
<tr>
<td>95°C</td>
<td>5min</td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>95°C</td>
<td>1min</td>
<td rowspan="3"><br />30 cycles<br /><br /></td>
</tr>
<tr>
<td>48°C</td>
<td>2min</td>
</tr>
<tr>
<td>72°C</td>
<td>3min</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>72°C</td>
<td>10min</td>
<td> </td>
</tr>
<tr>
<td>15°C</td>
<td>Hold</td>
<td> </td>
</tr>
</tbody>
</table>
<br /><br /><br /><br />

[STEPS]
TITLE: CMU Libraries Love Data Week 2020 - Valentine&#39;s Day Dessert Recipes
AUTHORS: Hannah Gunderman, Angelina Spotts, Emma Slayton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">At CMU Libraries, each year we celebrate Love Data Week (LDW) with events, workshops, speakers, and activities to help engage campus in the world of data that surrounds us! Similar to Open Access Week, the purpose of LDW events is to raise awareness and build a community to engage on topics related to research data management, sharing, preservation, reuse, and library-based research data services. We share practical tips, resources, and stories to help researchers at any stage in their career use good data practices. We believe research data are the foundation of the scholarly record and crucial for advancing our knowledge of the world around us.</div><div class = "text-block">CMU Libraries is proud to offer an institutional license to protocols.io, a collaborative platform for computational workflows, operational procedures, instructions/manuals, and more! It can even be used for cooking recipes! To highlight the research workflow capabilities of protocols.io, we are proud to offer several dessert recipes that are tried-and-true by our 2020 Love Data Week committee at CMU Libraries. Why not make them for a special someone this Valentine's Day?</div><div class = "text-block">We hope you enjoy, and feel free to contact us at UL-DataServices@andrew.cmu.edu if you'd like to use this platform for your research data needs!</div></div>

[STEPS]
TITLE: Morphometry of Ophiothrix (Echinodermata: Ophiuroidea)
AUTHORS: Renata Alitto, Pablo Damian Borges Guilherme, Michela Borges, Letícia de Oliveira Dias, Helena Serrano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Morphometry of </span><span style = "font-style:italic;">Ophiothrix </span><span>using PERMANOVA and LDA (Linear Discriminant)</span></div></div>

[STEPS]
?. [Measurements]
Measurements were taken using an ocular micrometer and through the AxioVision VS program 40.4.8.20 (Carl Zeiss Microscopy, Germany) attached to a ZEISS Discovery V20 stereomicroscope for specimens less than 10 mm disc diameter and with a digital Mitutoyo CD-6 CS caliper for the larger specimens.
?. [Characters]
Disc diameter, Radial shield length, Radial shield width, Disc spine, Arm spine, Second arm spine, Oral diameter, Oral shield length, Oral shield width, Adoral shield length, Adoral shield width, Dorsal arm plate length, Dorsal arm plate width,  1stventral arm plate length, 1stVentral arm plate width, 2ndventral arm plate length, 2ndventral arm plate width
?. [PERMANOVA]
A permutational multivariate analysis of variance (PERMANOVA) was conducted (function adonis) and homogeneity of group dispersions (functions betadisper and permutest) were then used to formally test whether morphological characters and dispersion differed between CS (package vegan).
?. [LDA]
The linear discriminant analysis (LDA) was applied using the R environment. To avoid multicollinearity among morphological characters a correlation matrix was constructed and the variables that were significantly correlated were removedwith a threshold value of 0.9. To investigate the differences in morphological characters,  lda function (package MASS) was used to distinguish the four Ophiothrix CS. The classification rate of each CS was assessed by the “lda” function. The predict function (package STATS) and table function (package BASE) were used to assess the classification based on the linear discriminants and to verify the model error. Visualization was performed using the package ggplot2.Ophiothrix angulata specimens from the type locality (USNM) were predicted by the model to verify the CS to which they were most related.
TITLE: Preparation of bacterial cell lysate for SDS-PAGE (2022 iGEM)
AUTHORS: Team Fudan iGEM

[DESCRIPTION]
Preparation of bacterial cell lysate for SDS-PAGE

[STEPS]
SECTION: Preparation of bacterial cell lysate for SDS-PAGE
1. Grow the cells overnight, to an OD600 above 1. If IPTG induction needed, at it around OD600 0.2-0.3 (keep IPTG stock solution at -20 freezer).
SECTION: Preparation of bacterial cell lysate for SDS-PAGE
2. Take one 1 ml of OD600 = 1 from each sample. Correct the sample volume to obtain an equivalent size pellet. For us, as quantified using NanoCym950 nanoparticles (equivalent to the size of E. coli), 1 OD600 equals to 10^8 nanoparticles per mL. We estimate each well on SDS-PAGE contains proteins from 2*10^6 bacteria.
SECTION: Preparation of bacterial cell lysate for SDS-PAGE
3. Spin down the bacterial cells for 1 min at 13,000 rpm in a microfuge.
SECTION: Preparation of bacterial cell lysate for SDS-PAGE
4. Aspirate any trace of supernatant with a vacuum line avoiding touching the pellet, but removing all liquid. 
SECTION: Preparation of bacterial cell lysate for SDS-PAGE
5. To each cell pellet, add 250 µL of distilled water, resuspend the pellet. Add 250 µL 2x SDS sample buffer (see below). Heat with a 95 degree heating block for 10 minutes. Store the samples at -20 degree freezer until SDS-PAGE.
SECTION: Receipt
6. 2x SDS sample buffer, works for Tris-Cl or MOPS based gels
Glycerol 20 ml
SDS 4 g
0.5 M Tris-HCl pH 6.8 stock solution 25 ml
Bromo Phenol Blue 1 mg; dissolve and volume to 95 ml
add 5% β-Mercaptoethanol before usage
TITLE: Protocol for Bulk Water Enrichment for the Isolation of Salmonella from Surface Water
AUTHORS: Autumn L Kraft, Manan   Sharma, Jonathan G Frye, James  E Wells, Abasiofiok  Mark Ibekwe, NARMS EWG  

[DESCRIPTION]
This protocol details the bulk water enrichment for the isolation of Salmonella from surface water.

[STEPS]
SECTION: Protocol
1. Take 1 of the water sample bottles (containing 1 L of surface water) and carefully transfer water into a sterile 2 L Pyrex bottle.

SECTION: Protocol
2. If needed, add control strain to water sample by gently tipping vial over the opening. Close bottle tightly and thoroughly mix sample.
SECTION: Protocol
3. Aseptically measure 250 mL of 5X UPB and pour into 2 L bottle (making final concentration of 1X UPB)

SECTION: Protocol
4. Secure lid and mix thoroughly before incubating at 37 °C for 18-24 h.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This short protocol describes how to maintain your fly stocks as single generation, age-matched populations. The method described encourages a health fly population, firstly, as individuals mate and oviposit during an optimal age when females are highly fecund and secondly, because the reduced crossing over of generations reduces the risk of horizontal disease<br />transmission.</p>

[STEPS]
?. 
?. 
?.
TITLE: GENOTYPING OF iPSCS WITH GENE INSERTIONS (Support Protocol 1)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will use genomic DNA isolated from the purified iPSC clones with CLYBL or AAVS1 gene insertion to determine if integration of the transgene has occurred correctly, in a heterozygous or homozygous fashion, and if the floxed selection genes are present. Primer sequences, amplicon sizes, PCR mix composition, and thermal conditions are included in the steps.</div></div>

[STEPS]
?. Set up PCR reaction (also see Kramer & Coen, 2001). For each 10 μl reaction, use the primer pairs recommended in Reagents and Solutions for the given safe-harbor site and donor construct chosen: Primer 1 Primer 2 purified genomic DNA from iPSC clone H2O 2 × PCR Master Mix.
1 µl
1 µl
1 µl
2 µl
5 µl
?. Run PCR reactions  AB195 °C3min295 °C30 sec364 °C for CLYBL primers, 58 °C for AAVS1 primers30 sec472 °C1min5Repeat (to step 2) 34 times672 °C5min7Hold at 12 °CIndefinitely
AB195 °C3min295 °C30 sec364 °C for CLYBL primers, 58 °C for AAVS1 primers30 sec472 °C1min5Repeat (to step 2) 34 times672 °C5min7Hold at 12 °CIndefinitely
?. Run PCR products on 1 % agarose gel (Voytas, 2001) with interpretation given above for each primer pair.
If performing the PCR on samples following Cre excision, increasing the amount of time during the extension step may be necessary to detect the larger un-excised template if a heterogeneous culture is obtained.
TITLE: UC Davis - Glugagon
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Glucagon is a 29 amino acid polypeptide processed from proglucagon in pancreatic alpha cells. In intestinal L-cells proglucagon is cleaved into glicentin, corresponding to proglucagon residues no 1-69. Glicentin can further be processed into oxyntomodulin, corresponding to proglucagon residues no 33-69. These peptides are released simultaneously upon stimulation. Moreover, a fragment of glucagon corresponding to its Cterminal part (residues no 19-29), also designated mini-glucagon, is reported to be present in the pancreas in low amounts compared to the total glucagon content. </div><div class = "text-block">In general, glucagon has an effect opposite that of insulin, i.e. it raises blood glucose levels. It causes the liver to convert glycogen into glucose, which is then released into the blood stream. With longer stimulation, glucagon action in the liver results in a glucose-sparing activation of free fatty acid oxidation and production of ketones. During hypoglycaemia, glucagon secretion offers a protective feedback mechanism, defending the organism against damaging effects of glucose deficiency in the brain and nerves.</div></div>

[STEPS]
?. Prepare enzyme conjugate 1X solution and wash buffer 1X solution.
?. Prepare sufficient microplate wells to accommodate Calibrators, controls and samples in duplicate.
?. Pipette 10 µL each of Calibrators, controls and samples into appropriate wells.
?. Add 50 µL enzyme conjugate 1X solution to each well and attach the plate sealer.
?. Incubate on a plate shaker (700-900 rpm) over night (18-22h) at 2–8ºC
?. Wash 6 times with 700 µL wash buffer 1X solution per well using an automatic plate washer with overflow-wash function. After final wash, invert and tap the plate firmly against absorbent paper. Do not include soak step in washing procedure.Or manually,Discard the reaction volume by inverting the microplate over a sink. Add 350 µL wash solution to each well. Discard the wash buffer 1X solution, tap firmly several times against absorbent paper to remove excess liquid. Repeat 5 times. Avoid prolonged soaking during washing procedure.
?. Add 200 µL Substrate TMB.
?. Incubate on the bench for 30 minutes at room temperature (18–25ºC).
?. Add 50 µL Stop Solution to each well. Place plate on a shaker for approximately 5 seconds to ensure mixing.
?. Read optical density at 450 nm and calculate results. Read within 30 minutes.