eamag/protocols-with-steps huggingface dataset:
TITLE: Endo H/ PNGase F Assay for JESS Automated Western Blot 
AUTHORS: Charis Ma, Yu Chen, sidranse

[DESCRIPTION]
This protocol details an optimized Endo H and PNGase F digestion assay that produces samples that can be run with the JESS Automated Western Blot System. 

[STEPS]
SECTION: Glycoprotein Denaturation
1. In an eppendorf tube, add 40 µg protein, 2 µL 10X Glycoprotein Denaturing Buffer, and water for a total reaction volume of 20 µL . Mix well.

SECTION: Glycoprotein Denaturation
2. Denature glycoprotein by heating reaction at 100 °C  for 10 min .

SECTION: Glycoprotein Denaturation
3. Split reaction volume into 2 eppendorf tubes with 10 uL in each tube. One tube will be used for the EndoH reaction, while the other will be used for the PNGaseF reaction.
SECTION: Endo H
4. Add the following to one reaction tube from step 3: 2 µL  10X GlycoBuffer 3, 2 µL Endo H, 6 µL water. Total volume will be 20 µL .

SECTION: Endo H
5. Mix gently.
SECTION: Endo H
6. Incubate reaction at 37 °C for 60 min

SECTION: PNGase F
7. Add the following to the second reaction tube from step 3: 2 µL 10X GlycoBuffer 2, 2 µL NP-40, 6 µL water, 1 µL PNGaseF. Total volume will be 21 µL .

SECTION: PNGase F
8. Mix gently.
SECTION: PNGase F
9. Incubate reaction at 37 °C for 60 min.

SECTION: Desalting and Washing for JESS Automated Western Blot System (Bio-Techne) preparation
10. Add 480 µL and 479 µL of 0.1X Sample Buffer (Bio-Techne) to the Endo H and PNGaseF reaction tubes respectively to make the final volume 500 µL.

SECTION: Desalting and Washing for JESS Automated Western Blot System (Bio-Techne) preparation
11. Add all 500 µL of each reaction to a 10K Amicon Ultra Centrifugal Filter 

SECTION: Desalting and Washing for JESS Automated Western Blot System (Bio-Techne) preparation
12. Spin at 14000 x g for 20 min. You will recover a final volume of20 µL, and the protein concentration recovered will be ~1 mg/mL .

SECTION: JESS Sample Preparation
13. Add 4 parts of the washed Endo H/ PNGaseF sample from step 12 with 1 part of 5X Fluorescent Master Mix (Bio-Techne). 
SECTION: JESS Sample Preparation
14. Vortex to mix
SECTION: JESS Sample Preparation
15. Incubate sample at 95 °C for 5 min.

SECTION: JESS Sample Preparation
16. Load into JESS microplate (Bio-techne) to be detected by the Automated Western Blot System.
TITLE: 0.1% Cresyl Violet Stain (pH 4.3)
AUTHORS: Shaina Robbins, Sean Nieves

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

[STEPS]
?. [Solution Prep]
Measure  of ddH2O or molecular grade H2O and pour into a glass beaker
500 ml
?. [Solution Prep]
Aliquot  of cresyl violet acetate into the glass beaker from the above step
0.5 g
Causes skin irritationMay cause respiratory irritationCauses serious eye irritation
?. [Solution Prep]
Put a magnetic stir bar into the glass beaker (containing H2O and cresyl violet acetate) and place on a hot plate at and stir until cresyl violet is dissolved
100 °C
Allow solution to come to room temperature once cresyl violet is dissolved
?. [pH]
Test the pH of the solution. Add glacial acetic acid dropwise until the desired (pH 4.3) is achieved
?. [Filter]
Pour the contents of the 0.1% Cresyl Violet solution through a sterile membrane filter into a light sensitive jar and close lid
TITLE: Phage lambda genome concatemerization
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to create concatemers of phage genomes. This is done by ligating phage lambda genomes using the overhangs at the cos site with T4 ligase. </div></div>

[STEPS]
?. Add the following ingredients in a microcentrifuge tube.  AB1Ingredient&nbsp;Amount&nbsp;2Ligation buffer 10X&nbsp;2&nbsp;µL&nbsp;3DNA&nbsp;&nbsp;6&nbsp;µg&nbsp;4PEG 4000&nbsp;(40% W/W)&nbsp;2.5&nbsp;µL&nbsp;5T4 DNA ligase&nbsp;1&nbsp;µ&nbsp;L&nbsp;(400 Weiss units)&nbsp;6Water&nbsp;To 20&nbsp;µL&nbsp;
AB1Ingredient&nbsp;Amount&nbsp;2Ligation buffer 10X&nbsp;2&nbsp;µL&nbsp;3DNA&nbsp;&nbsp;6&nbsp;µg&nbsp;4PEG 4000&nbsp;(40% W/W)&nbsp;2.5&nbsp;µL&nbsp;5T4 DNA ligase&nbsp;1&nbsp;µ&nbsp;L&nbsp;(400 Weiss units)&nbsp;6Water&nbsp;To 20&nbsp;µL&nbsp;
?. Incubate at  overnight
15 °C
TITLE: Ketosium XS Keto Reviews : How To Use It?
AUTHORS: ketosiumketoreviews  

[DESCRIPTION]
Ketosium XS Keto Reviews : How To Use It?


[STEPS]
1. Ketosium XS Keto Reviews : How To Use It?
Are you floundering with weight problems and facing health problems, including feeling bad and losing energy and vitality? Do you no longer feel at ease with your standard of living as your energy, health, and vitality dwindle, and want to restore your sound health and vibrant life? Is hunger an issue for you, and do you constantly worry for fried and fattening food? Are you eager to find a important strategy that can help you lose all the redundant weight fluently? 

Also, we've Ketosium XS Keto available for you through which the problem of hunger will be reduced, and you'll eat only healthy food and ameliorate your impunity and stamina. This formula controls your sugar position and helps control your body weight. This chemical-free product has numerous advantages and you can use this product by following the necessary directions. You must review the details in the composition about the product. 

About Ketosium XS Keto 

Ketosium XS Keto is a salutary supplement that's designed to help people lose weight. The product is made with ketone mariners, which are allowed to help the body burn more fat. Ketosium XS Keto is also claimed to suppress appetite and boost energy situations. Some exploration suggests that ketone mariners may be salutary for weight loss, but further studies are demanded. 

Still, your metabolism position may be poor, your impunity may be low power, If you're fat and have colorful health issues. These health issues impact numerous people currently. Every person is defying these medical conditions every second. Ketosium XS Keto is concentrated on helping you as it's designed to exclude all of the redundant fat from your system, makes you more energetic and active, and improves your core values. 

How Does Ketosium XS Keto Work? 

Ketosium XS Keto is a salutary supplement that's designed to help people lose weight. The product contains ketones, which are substances that are produced when the body breaks down fat for energy. Ketosium XS Keto is said to help the body burn further calories and lose weight briskly. 


Official Website:- https://freaknewswire.com/index.php/2022/05/06/ketosium-xs-keto-reviews-where-to-buy/
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for separating total RNA using denaturing formaldehyde agarose gel electrophoresis.</p>
<p>This method can be used to separate larger RNAs in a range of 400-6000 nt, either for quality control or downstream Northern Blot Analysis.</p>
<p> </p>

[GUIDELINES]
<p>Always wear gloves when handling hazardous materials.</p>
<p>Always handle RNA cold and in an RNase free manner.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Cytoplasmic pH measurements with pHluorin 
AUTHORS: Ekaterina Krasnopeeva, Teuta Pilizota

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Single-cell </span><span style = "font-style:italic;">in vivo </span><span>cytoplasmic pH measurments with genetically encoded pHluorin sensor</span></div></div>

[STEPS]
?. [Sample preparation]
Grow cells to the desired OD according to a chosen growth protocol.
?. [Sample preparation]
Prepare a tunnel-slide/flow cell and attach cells to the coverslip with the preferred surface attachment method (Wang et al. 2019)
It is preferable to use a non-fluorescent medium for imaging. If needed you can clean your coverslips with KOH or by baking them.
?. [Cells imaging]
Cell imaging is done by custom-made microscope with Nikon CFI Plan ApochromatLambda 100x objective (NA 1.45) and iXon Ultra 897 EMCCD camera. Narrow spectrum 395 nm LED is used for the excitation at 395 nm and Neutral white LED with ET470/40x filter for 475 nm excitation. Emission is collected at 520 nm using ET525/40m filter.Imaging settings vary depending on the experiment.
?. [pH sensor calibration]
Perform the pH sensor calibration using the same conditions and imaging parameters as the main experiment.Supplement experimental medium with pH collapsing agent PBMH (40 mM potassium benzoate and 40 mM methylamine hydrochloride) and adjust its pH to the range of values between 5.5 and 9.
?. [pH sensor calibration]
Flush the experimental medium of known pH into a sample chamber (when using a tunnel slide we wick it into the tunnel with a pipette at one entrance of the channel and a small tissue on the other), incubate for 15~min. Take images of 5-10 different fields of view containing at least 100 cells in total with the same imaging parameters as the main experiment.
?. [pH sensor calibration]
Plot the calibration curve as a ratio of emission intensities for excitation at 395 nm and 475 nm against pH.Fit with the sigmoid function R395/475=(a1 ek(pH-pH0)+a2)/(ek(pH-pH0)+1), where a1, a2, k and pH0 are free fitting parameters.Use the calibration curve to recover pH from intensity ratio data.
?. [pH sensor calibration]
Analyse images recovering emission intensities for 395 and 475 nm excitation channels. We use custom-written Python script to calculate mean intensities of the single cells' cytoplasm fluorescence.
TITLE: LB Broth
AUTHORS: sgoodwin

[DESCRIPTION]
Process for producing LB broth growth medium

[STEPS]
SECTION: Making the LB broth
1. Dilute 20 g of LB powder in 800 mL distilled water

SECTION: Making the LB broth
2. Stir until fully dissolved with magnetic stirrer
SECTION: Making the LB broth
3. Transfer to a Duran flask, and sterilise by autoclaving
TITLE: Radioactive Labeling with T4 PNK (M0201)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Radioactive Labeling with T4 Polynucleotide Kinase.

[STEPS]
1. Set-up the following reaction:  
 

 

2. Incubate at 37 °C for 30 min.  
3. Heat inactivate by incubating at 65 °C for 20 min.
TITLE: Nissl Counterstaining - Cresyl Violet
AUTHORS: Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Nissl Counterstaining with Cresyl Violet for histological sections 

[STEPS]
SECTION: Delipidation Step
1. Ethanol 95%, 5min
SECTION: Cresyl Violet staining
7. Cresyl Violet solution, 1min
SECTION: Cresyl Violet staining
8. dH2O, 15sec or less (quick rinse)
SECTION: Cresyl Violet staining
9. dH2O, 2min
SECTION: Differentiation
10. Ethanol 70%+Acid Acetic*, few seconds (control differentiation yourself). (*add 2-3 drops
of acetic acid 100% in 100ml ethanol 70%).
SECTION: Dehydratation
11. Ethanol 95%, 5-10sec (quick rinse)
SECTION: Dehydratation
12. Ethanol 100%, 1min
SECTION: Dehydratation
13. Xylene, 1min or more (under the hood) until mounting
SECTION: Dehydratation
14. Mount coverslip with DPX (under the hood)
SECTION: Delipidation Step
4. Ethanol 95%, 2min
SECTION: Delipidation Step
3. Ethanol 95%, 15 sec (quick rinse)
SECTION: Delipidation Step
2. Chloroform 100%, 10min (under the hood)
SECTION: Delipidation Step
5. Ethanol 70%, 1min30sec
SECTION: Delipidation Step
6. dH2O, 2min
TITLE: H&amp;E staining on paraffin sections
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes H&E staining on fly heads paraffin sections for neuronal counting experiments.

[STEPS]
SECTION: For neuronal counting experiments
1. Fix fly heads   in 4 %.
SECTION: For neuronal counting experiments
2. Embed in paraffin.
SECTION: For neuronal counting experiments
3. Cut 4 micron sections.
SECTION: For neuronal counting experiments
4. Dry at 42 °C  .
SECTION: For neuronal counting experiments
5. Deparaffinize and bring through ethanols to water (xylene x 2, 100 % x 4, tap H2O x 2).
SECTION: For neuronal counting experiments
6. Stain with Harris’ hematoxylin for 10 min .
SECTION: For neuronal counting experiments
7. Rinse 3x tap H2O.
SECTION: For neuronal counting experiments
8. Differentiate in acid alcohol (1 % in 70 %) – one quick dip.
SECTION: For neuronal counting experiments
9. Rinse 3x tap H2O.
SECTION: For neuronal counting experiments
10. Blue in tap H2O for 5 min  or to desired color.
SECTION: For neuronal counting experiments
11. Rinse 3x tap H2O.
SECTION: For neuronal counting experiments
12. Stain with eosin (10 % in 100 %) – one quick dip or to desired color.
SECTION: For neuronal counting experiments
13. Rinse 2x 100 %.
SECTION: For neuronal counting experiments
14. Dehydrate through ethanol series and xylenes.
SECTION: For neuronal counting experiments
15. Mount with Permount.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Validated with : rodent liver, kidney, eye, skeletal muscles, adrenal gland. </p>

[BEFORE_START]
<ol>
<li>Prepare the lysis buffer on the day of the extraction by adding 1% of beta-mercaptoethanol to the total needed RLT volume.</li>
<li>Use 1000ul of RLT+1% β-ME per sample if starting with the suggested amount of 20+/-5mg of tissue (Muscles&lt;20mg, all other tissues &lt;/=30mg) </li>
<li>Confirm that ethanol was added to buffer RPE, AW1, AW2</li>
<li>Prepare the DNase right before the digestion step. </li>
<li>Prepare 2 buckets of wet ice : for RNA samples post trap-on-column step and for tissue lysate and DNA samples. </li>
</ol>

[GUIDELINES]
<ol>
<li>Make sure to seperate RNA and DNA work</li>
<li>Work quickly and efficiantly to preved RNA degradation</li>
<li>Make sure to not thaw the tissues at the time of weighting, work on dry ice. </li>
<li>Stopping point at DNase digestion step</li>
<li>At the time of the homogenization, keep the samples on wet ice </li>
</ol>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Endocytosis and internalization assay in primary neuronal culture
AUTHORS: Arpine Sokratian, andrew.west west

[DESCRIPTION]
This protocol outlines a method for quantifying endocytosis in primary neuronal cultures, specifically focusing on different a-synuclein fibril structures. By utilizing pH-sensitive dye conjugated to experimental proteins/fibrils, we can identify endocytosis pathways and analyze intensity changes in the pHrodo-specific channel from real-time acquired images at various time points. 

[STEPS]
SECTION: Preparation of endocytosis inhibitors
2. Prepare stock solution of the endocytosis inhibitors in DMSO: 
 
 

SECTION: Preparation of endocytosis inhibitors
3. At the day of the experiment, thaw down the stock solutions in a water bath and dilute the drugs to reach concentrations: 



 

SECTION: Preparation of endocytosis inhibitors
1. For the assay we use DIV7 primary hippocampal neuron culture plated in 48-well plates
SECTION: Preparation of endocytosis inhibitors
4. Before adding protein/drug of interest, add endocytosis inhibitors 30 minutes before the treatment. As calculated for 48-well plates with 3 mL of the media, 30 ul of diluted drugs are sufficient. ** Note: Be careful adding the diluted drugs (should be RT). Place the plate back to the cell culture incubator.
SECTION: Preparation of endocytosis inhibitors
5. After 30 minutes, add the protein/drug of interest supplement with   
As for phRodo-conjugated fibrils, please, see the protocol describing the conjugation process (step 5): 
As a control for endocytosis add lysotracker   and phrodo-10kDA dextran   

 




SECTION: Preparation of endocytosis inhibitors
6. After two hours of incubation, start taking images of each well at the high-speed (high sensitive mode)
Continue acquiring images every two to four hours up to 48 hours of incubation. 
SECTION: Preparation of endocytosis inhibitors
7. Calculate the images using cell count (Hoechst) for normalization and calculate the signal for each time-point as a proportion to the maxima of the signal 

Calculate the signal from Lysotracker after 30 minutes of incubation and compare the control (no endocytosis inhibitors) to the experimental reactions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A set of Stellaris FISH Probes comprises up to 48 singly labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. Please see the Biosearch

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Dear researcher,</p>
<p>This protocol gives you the steps to start an MRI project at the Spinoza Centre Roeterseiland. </p>
<p>If you haven't had contact with Steven Scholte or Tinka Beemsterboer, make sure to contact them before completing this protocol. </p>
<p> </p>
<p>You can reach us by email:</p>
<p>Steven Scholte: h.s.scholte@uva.nl</p>
<p>Tinka Beemsterboer: t.beemsterboer@uva.nl</p>
<p> </p>
<p>If you have any suggestions for improving the workflow, don't hesitate to add comments to the steps in the protocol.</p>
<p>Hope to see you soon!</p>
<p> </p>
<p>Spinoza REC Team!</p>
<p>Steven Scholte, Tinka Beemsterboer en Lukas Snoek </p>
<p> </p>

[STEPS]
?. 
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?.
TITLE: Sequencing for Bark beetles and associated fungi
AUTHORS: Jiri Hulcr, Sawyer Adams

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to describe the submission for sequencing at the UF Forest Entomology Lab.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: ELISA
AUTHORS: Pranay Srivastava

[DESCRIPTION]
This protocol is to determine the levels of IL-1β and TNF-α in plasma and brain (Srivastava et al. 2020).

[STEPS]
1. Blood samples from all the treatment groups were collected through cardiac puncture in 40 mM EDTA and plasma was collected by centrifugation. 
2. The plasma samples were immediately transferred to dry ice and then stored at -80°C till further analysis. 
3.  A small fraction of ventral midbrain tissue homogenate prepared in 1x RIPA buffer (Cell Signaling, Cat#9806) was used for analysis of IL-1β and TNF-α using mouse ELISA kits (Biolegend Cat# 430904; 432604).
TITLE: Imaging Mass Cytometry Compensation Slide Preparation
AUTHORS: Michelle Daniel, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes the preparation of compensation slides with single antibodyconjugate</div><div class = "text-block">spots that can be used to estimate mass channel spillover for IMC. Spillover</div><div class = "text-block">varies from metal lot to lot due to varying impurities. Once a spillover matrix for a specific</div><div class = "text-block">set of antibody-conjugates has been generated, the experiment of interest, in which the</div><div class = "text-block">same antibody conjugates have been used, can be spillover-corrected.</div><div class = "text-block">It is crucial that this procedure is performed for the exact same antibody conjugates that</div><div class = "text-block">are used to pipette the antibody panel for sample staining.</div><div class = "text-block">The original publication can be viewed here:</div><div class = "text-block">“Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry” by S.</div><div class = "text-block">Chevrier, HL Crowell and VRT Zanotelli et al., Cell Systems, 2018</div></div>

[STEPS]
?. [Generation of agarose coated slides]
1. Prepare 2% agarose in ddH2O in small beaker and melt, e.g. in a microwave.2. Preheat microscopy slide to 70°C on a hot plate.3. Distribute ca. 350-400 μl of liquid agarose over the slide until it is covered.4. Let the agarose dry for at least 30 min.
At the end of this section the agarose coat will be very thin, homogeneous and barely visible.These slides can be stored until further usage at room temperature in dry conditions.
?. [Generation of antibody conjugate dilutions]
1. Pipette an array of 0.3 μl trypan blue spots onto the agarose coated slide. The spots should bewell separated from each other. Prepare as many trypan blue spots as the number of antibodyconjugatedmass-channels used in the study to be compensated (e.g. for every antibody).2. In AirLab open the relevant panel and print it out.3. Retrieve all listed antibody conjugates from the 4 °C fridge and keep them on ice.4. For each antibody conjugate, pipette 0.2 μL onto the trypan blue spot on the agarose coatedslide. Note the exact location of each conjugate on the slide and make sure that no material bleedover occurs. For simplicity, it may be preferable to sort the antibodies according to the mass ofthe conjugated metal.5. Let the slide dry for at least 1h.6. Store the slide at room temperature in a dedicated box for measurement.
In this step the metal spots are created on the agarose coated slide. The metal is diluted in theblue organic dye “trypan blue”. This should help to locate the metal spots on the spillover slide.
TITLE: Quality Assurance and Quality Control (Part 10 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 10 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:        ELISA for quantification of Granulocyte-colony stimulator factor (G-CSF) in human serum or plasma
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human granulocyte-colony stimulator factor (G-CSF) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human G-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-G-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-human G-CSF antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. 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 G-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 G-CSF standard dilutions and the human G-CSF sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.