eamag/protocols-with-steps huggingface dataset:
TITLE: Cell Surface Mild Acid Elution of MHC-bound Immunopeptides
AUTHORS: Teesha Luehr

[STEPS]
?. [Culturing Cells]
Culture cells. If culturing THP-1s, follow the protocol.
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?. [Culturing Cells]
Grow cells to a total number of 1-2x10*8 cells
?. [Preparing Reagents]
1X PBS at
[(room temperature)]
[NaCl]
[KCl]
[NA2HPO4]
[KH2PO4]
[Milli-Q water]
?. [Preparing Reagents]
1X PBS at
4 °C
This PBS solution does NOT have to be sterile, but make sure it is clearly marked.
?. [Preparing Reagents]
1X saline (PBS without the phosphate) at no need to pH adjust, it's not a bufferno need to sterilize as the mild acid elution is not done under sterile conditions
4 °C
[NaCl]
[KCl]
[Milli-Q water]
?. [Preparing Reagents]
1X saline + 2% acetic acid at
4 °C
[of above 1X saline solution]
[acetic acid]
?. [Pelleting & Rinsing Cells]
Cells will most likely be in . Use five to pellet all cells from one flask at a time.
[complete media]
[conical tubes]
?. [Pelleting & Rinsing Cells]
Pellet cells for
[at 500g]
?. [Pelleting & Rinsing Cells]
Resuspend each pellet by pipetting up and down 5X with
[room temp PBS (sterile in the hood)]
?. [Pelleting & Rinsing Cells]
Combine all five conical tubes into one
?. [Pelleting & Rinsing Cells]
If cell count is not know, perform one now to confrim a minimum of 1x10*8 cells
?. [Pelleting & Rinsing Cells]
Pellet cells for
[at 500g]
?. [Pelleting & Rinsing Cells]
Discard supernatant
?. [Pelleting & Rinsing Cells]
Discard supernatant
?. [Pelleting & Rinsing Cells]
Resuspend each pellet by pipetting up and down 5X with
[cold PBS]
?. [Pelleting & Rinsing Cells]
Pellet cells for
[at 500g]
?. [Pelleting & Rinsing Cells]
Discard supernatant
?. [Pelleting & Rinsing Cells]
Resuspend each pellet by pipetting up and down 5X with
[cold PBS]
?. [Pelleting & Rinsing Cells]
Pellet cells for
[at 500g]
?. [Pelleting & Rinsing Cells]
Discard supernatant
?. [Pelleting & Rinsing Cells]
Resuspend each pellet by pipetting up and down 5X with
[cold saline]
?. [Pelleting & Rinsing Cells]
Pellet cells for
[at 500g]
?. [Pelleting & Rinsing Cells]
Discard supernatant
?. [Mild Acid Elution]
Resuspend each pellet by pipetting up and down 5X with
[cold 2% acetic acid in saline]
EXTREMELY IMPORTANT, REMEMBER THAT THE PEPTIDES WILL BE IN THE SUPERNATANT
?. [Mild Acid Elution]
DO NOT DISCARD SUPERNATANT
?. [Mild Acid Elution]
Collect the supernatant in a new
[conical tube]
?. [Pelleting & Rinsing Cells]
Transfer cells + saline to a new
[conical tube]
This is to remove any large amounts of phosphate
?. [Preparing Reagents]
Each will require 7
[T-175 flask]
[conical tubes]
?. [Drying]
Freeze supernatant at
[overnight]
?. [Drying]
Lyophilize sample for 48 hours
?. [Drying]
Peptides are now ready to be desalted with chosen clean up method (STAGE tip, LC-UV, combination, etc)
TITLE: Soil Organic Carbon Stocks and Change
AUTHORS: SCarolina Córdova, Curtis Dell, Mark Liebig, michel.cavigelli, Phil Robertson

[DESCRIPTION]
The change in soil organic carbon (SOC) accumulation or decreases over time serves as an integrated indicator of carbon (C) balance in cropping systems, and as an integral metric of soil health assessments that can be carried out at all LTAR locations. While measurement of C flux with micro-metrological methods, such as eddy-covariance, provides very precise measurements of CO2 flux into and out of ecosystems, measurement systems are expensive, require a high-level of expertise to operate, and are not applicable for plot-scale measurements.Determination of SOC concentration and soil bulk density allows calculation of the mass of SOC accumulated in the soil, and changes in SOC over time reflect system C balance. In most soils measurable SOC changes require a minimum sampling interval of 10 to 12 years depending on local factors like existing SOC stores and rotation diversity. Thus, we recommend deep soil cores to 1 m depth taken at decadal intervals, and in intervening years (e.g., five-year intervals), surface soil samples at 0-25 cm or 0-30 cm depth, which includes the most active part of the rootzone. In cropping systems sampling intervals should be timed to the same crop phase and at the same time of year to minimize year-to-year variability in root and residue inputs; sampling post-harvest can minimize plant disturbance. A dry combustion CN analyzer should be used to measure total C; for soils with detectable carbonate minerals, inorganic C must first be removed by acid fumigation or analyzed independently for later subtraction. Estimation of SOC stocks can be calculated using two methods: spatial coordinate (includes bulk density), and equivalent soil mass (normalized mass per unit area). 

[STEPS]
TITLE: XPRIZE SHINE - In-tube Fluorescent SARS-CoV-2 Saliva Test
AUTHORS: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to perform a SHINE in-tube fluorescent assay to detect SARS-CoV-2 RNA from a self-collected nasopharyngeal sample. This protocol is intended for point-of-care use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol requires a transilluminator or another equivalent blue light emitting device. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, </span><span style = "font-style:italic;">et al.</span><span> Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. </span><span style = "font-style:italic;">bioRxiv</span><span> (2020). </span></div></div>

[STEPS]
?. [Sample Collection and Viral Lysis]
Expel approximately one drop of saliva into the sample collection tube and cap the tube.    Saliva collection tube contains necessary volume of FastAmp® Viral and Cell Solution.
?. [Sample Collection and Viral Lysis]
Mix saliva sample and FastAmp® Viral and Cell Solution by vortexing the closed sample collection tube for .
?. [Sample Collection and Viral Lysis]
Wait , incubating sample at , before proceeding to Step 4.
0 Room temperature
?. [SARS-CoV-2 Detection]
Pipette  of Reagent Mix A into a single uncapped well of the 96-well plate containing lyophilized Reagent Mix B. Mix by pipetting up and down gently.
15 µl
?. [SARS-CoV-2 Detection]
Add  sample-viral lysis mix to Reagent Mix A and B well. Mix by pipetting up and down gently. Recap sample.
5 µl
?. [SARS-CoV-2 Detection]
Wait  , incubating sample at , before proceeding to Step 7.
0 Room temperature
?. [In-tube Fluorescent Readout and Automated Analysis]
Visualize the fluorescence of the sample using a transilluminator or equivalent blue light emitting device.
?. [In-tube Fluorescent Readout and Automated Analysis]
With the user-provided smart device such as a smartphone, open the HandLens application and select in-tube as the test type.
?. [In-tube Fluorescent Readout and Automated Analysis]
Take a photo of the plate, and select upload. The result of the test will appear on the smart device screen.
TITLE: Recombinant a-synuclein pre-formed fibril generation
AUTHORS: Alain Ndayisaba

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the generation of a-synuclein pre-formed fibrils.</div></div>

[STEPS]
?. [Recombinant a-synuclein pre-formed fibril generation]
Reconstitute  of lyophilized monomeric synuclein with  cold sterile DPBS  (do not pipet, close tube immediately).
1 mg
100 µl
on ice
?. [Recombinant a-synuclein pre-formed fibril generation]
Transfer tubes on wet ice to  cold room and rotate on tube rotator for .
4 °C
?. [Recombinant a-synuclein pre-formed fibril generation]
Centrifuge for  at  at .
Centrifuge: 15000 34
4 °C
?. [Recombinant a-synuclein pre-formed fibril generation]
Transfer supernatant to clean Protein-LoBind tube.
?. [Recombinant a-synuclein pre-formed fibril generation]
Prepare  aliquots of serial dilutions (1:10, 1:25, 1:50, 1:100) and measure concentration with Nanodrop (A280 - MW=14,5 kDa; Extinction coefficient ε for human α-syn = 5,960 M-1cm-1).
5 µl
?. [Recombinant a-synuclein pre-formed fibril generation]
Dilute down to final concentration of , use  (-) aliquots for reproducible results.
100 µl
100 µl
500 µl
?. [Recombinant a-synuclein pre-formed fibril generation]
Take - aliquot of monomer and dilute to  for electron microscopy, flash-freeze in dry ice – ethanol slurry.
1 µl
2 µl
?. [Recombinant a-synuclein pre-formed fibril generation]
Seal tubes with parafilm if no tube lock.
?. [Recombinant a-synuclein pre-formed fibril generation]
Place the tube in an orbital thermomixer with a heated lid for  at , shaking at .
37 °C
Centrifuge: 1000 33
NOTE: At the end of the 7 days, the contents of the tube should appear turbid. The thermomixer must have a lid to prevent condensation formation on the tube lids.
?. [Recombinant a-synuclein pre-formed fibril generation]
Gently flick the tube to resuspend a-syn fibrils.a. Fibrils for quality control steps can be stored at .
37 Room temperature
?. [Recombinant a-synuclein pre-formed fibril generation]
Prepare aliquots for QC assays:
?. [Recombinant a-synuclein pre-formed fibril generation]
For long-term storage (12-18 months), flash-freeze fibrils in liquid nitrogen and store at .
-80 °C
?. [Recombinant a-synuclein pre-formed fibril generation]
a.  – thioflavin T binding assay.
5 µl
?. [Recombinant a-synuclein pre-formed fibril generation]
b.  for TEM.
2 µl
TITLE: Detection of bacteria in antibiotic-treated diatom cultures and cell harvesting
AUTHORS: Francesco Manfellotto, Monia Teresa Russo, Pina Marotta, Antonella Ruggiero, Mariella Ferrante

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We illustrate a simple and rapid method for detecting the presence/absence of bacteria in diatom cultures using DAPI and fluorescence microscopy. </div><div class = "text-block">We also describe how we harvest cells for genomic DNA extraction once we have assessed that bacteria are absent from the culture.</div></div>

[STEPS]
?. For the antibiotic treatment to obtain axenic cultures, see the published protocol:  Axenic Diatoms cultures protocoldx.doi.org/10.17504/protocols.io.bgudjws6Transfer 1 mL of Culture in a new 2 mL tube.
?. Add 1,6% of  neutralized formaldehyde to fix cells and mix gently by inversion
?. Add 1uL of DAPI 1 mg/mL and mix gently by inversion. Keep away from direct light
?. Transfer cells in a 35mm glass bottom dish.Incubate for 10 min away from direct light  to perimt cells sedimentation
?. Use an inverted microscope with fluorescence excitation. * We use a Leica DMI 6000 B microscope.Set light sources:bright-field  Fluorescence for chlorophyll (580 / 604 nm)Fluorescence for DAPI (364 / 454 nm)
?. Assess bacteria absence-presence in the cultures. Move across the plate, focusing on the cells and on the bottom of the dish.
?. Capture different photos
?. [Cells filtration]
Filter cells on MF-Millipore™ Membrane Filter, 1.2 µm pore size, gridded 47 mm.
?. [Cells filtration]
Move the filter in a new 50 mL tube using tweezers
?. [Cells filtration]
Rinse the filter with 1 mL of clean medium using a pasteur pipette pipet gently, to avoid cell lysis
?. [Cells filtration]
Collect the resuspended cells in a new 15 mL tube
?. [Cells filtration]
Pellet cells by centrifugation 15 min. 2000 g 18°C and discard supernatant
?. [Cells filtration]
Freeze pellet immediately in liquid nitrogen
?. [Cells filtration]
Store at -80°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.</span></p>
<p> </p>
<p><span style="font-weight: 400;">This protocol describes the steps for performing ICC/IF.</span></p>

[GUIDELINES]
<p>Tissue preparation is a key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, the protocol given here is intended as a starting point from which the experimenter must optimize as needed. All conditions should be standardized in order to ensure reproducible results. Keep in mind that you must be careful not to allow tissues to dry out at any time.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: DNA Cloning (Gibson Assembly, Transformation, Plating and Incubation)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM Team followed this protocol to construct the plasmid of interest or to clone the plasmid of interest.

[STEPS]
SECTION: Gibson Assembly
1. Calculate the volumes of respective DNA fragments to assemble based on their length and concentration. (The maximum final volume of the mixed fragments is 5 µL for each reaction.) 

SECTION: Gibson Assembly
2. Add the DNA fragments into a PCR tube according to the volumes obtained from the calculation.

SECTION: Gibson Assembly
3. Add 5 µL of NEBuilder HiFi DNA Assembly Master Mix into the same PCR tube.

SECTION: Plating and Incubation
19. Prepare an LB agar plate with the correct antibiotics.
SECTION: Plating and Incubation
20. Remove 950μL of the LB solution from the Eppendorf tube that contains the cell pellet, leaving about 100μL in the Eppendorf tube.
SECTION: Plating and Incubation
21. Resuspend the cells by pipetting the solution.
SECTION: Gibson Assembly
4. Vortex to mix the solution and centrifuge the PCR tube to spin down the remaining solution on the wall.
SECTION: Gibson Assembly
5. Incubate the PCR tube at 50°C for 60 min.

SECTION: Transformation
8. Take an Eppendorf tube that contains pre-made competent cells from the -80°C fridge.

SECTION: Transformation
9. Immediately place the Eppendorf tube with competent cells into the ice box for 5 min.

SECTION: Transformation
10. Add 20 µL  of the Gibson Assembly product or 1 µL of pure DNA plasmid into the Eppendorf tube containing the competent cells.

SECTION: Transformation
11. Tap the bottom of the Eppendorf tube to mix the solution.
SECTION: Transformation
12. Leave the Eppendorf tube in ice for 10 min.

SECTION: Transformation
13. Place the Eppendoft tube into a foam floating.
SECTION: Transformation
14. Place them into the water bath for 45 s at 42°C for heat shock.

SECTION: Transformation
15. Place the Eppendorf tube into the ice immediately.
SECTION: Transformation
16. Add 1 mL of the LB  media into the Eppendorf tube.

SECTION: Transformation
17. Place the Eppendoft tube into the incubator at 37°C for 60 min for recovery.

SECTION: Transformation
18. Centrifuge the Eppendorf tube to form a cell pellet (no specific speed and time).
SECTION: Transformation
6. Switch on the water bath and set the temperature to 42°C.
SECTION: Transformation
7. Prepare a box of ice.
SECTION: Plating and Incubation
22. Spread the cells onto the agar with the L-spreader. 
SECTION: Plating and Incubation
23. Place the petri dish in the incubator at 37°C for 60 min to allow the colonies to grow.
TITLE: Set Up Biodata Resource Inventory in Google Colab
AUTHORS: Kenneth Schackart

[DESCRIPTION]
This protocol will guide you on how to get everything in place to update the Biodata Resource Inventory.

This protocol describes how to setup Google Colab, connect your Google Drive, and clone the repository.

Some of the information here came from the following article: https://medium.com/analytics-vidhya/how-to-use-google-colab-with-github-via-google-drive-68efb23a42d

[STEPS]
SECTION: Prepare Google Drive
1. In your Google Drive home directory, create a new folder called GitHub.
SECTION: Connect Colab to Drive
2. Go to Google Colab.

If you need to change the account you are using, close the pop up by clicking Cancel at the bottom right. Change your Google account by clicking the icon at the top right.
SECTION: Connect Colab to Drive
3. A menu will pop up. At the bottom right, select: "New Notebook"
SECTION: Connect Colab to Drive
4. In the cell, paste or type the following:

 
Click the "run" button to the left of the cell or run with ctrl+enter

You will be prompted to allow Google Colab to access your Drive, say yes.
 

SECTION: Connect Colab to Drive
5. Create a new code chunk by clicking "+ Code" under the top-left banner. In that cell paste or type the following, then run:

 

This has changed your current directory to GitHub.
 

SECTION: Create GitHub Access Token
6. Go to GitHub.

Go to Settings, navigate to Developer settings and click on Personal access tokens.
SECTION: Create GitHub Access Token
7. Click Generate new token button on the top right corner of the page.

SECTION: Create GitHub Access Token
8. Click the repo checkbox under Select scopes.
SECTION: Create GitHub Access Token
9. Click the Generate token button at the bottom of the page.
SECTION: Create GitHub Access Token
10. You have now created a new access token. Copy the token to your clipboard. Do not close this page yet.

 

SECTION: Clone the Repository
11. Go back to your Colab Notebook, create a new Code cell, and enter the following, pasting your access token into the quotations, then run:

 

SECTION: Clone the Repository
12. Create a new code cell and paste or type the following, then run:
 

And cd into the repository:

 

Note: During development, the main branch is mostly empty. To switch to the development branch run the following in a code cell:

 

Congrats! You now have linked Google Colab to GitHub for access to their API, and you have cloned the repository into your Google Drive. You can now close the page with your access token.
SECTION: Running the pipelines
13. You can now access all the files in the repository via traditional Google Drive.
SECTION: Running the pipelines
14. You will find two notebooks in the repository:
running_pipeline.ipynb: This allows reproduction of the original results
updating_inventory.ipynb: This guides you to update the inventory

Each notebook provides instructions on their respective process, and includes already filled-in code chunks. So you should have to do little more work to run those pipelines.
TITLE: Effects of Caloric Restriction Associated with Low-Impact Aerobics and Strength Training Exercises on Body Compositions and Metabolic Syndrome Components among Obese students
AUTHORS: Mohamed Ahmed Said, Mohamed Abdelmoneem, Mohamed Chaab Alibrahim, Moustafa Ahmed Elsebee, Ahmed Abdel Hamed Kotb.

[DESCRIPTION]
Diet and physical activity are the most commonly recommended strategies for preventing and treating obesity and metabolic conditions. This study aims to examine the effectiveness of a weight reduction intervention based on caloric restriction, low-impact aerobics (LIA), and a resistance-training program in improving patients' anthropometric and biochemical indicators and the incidence of metabolic syndrome (MetS) among youth with obesity. In all, 64 participants were introduced to an enhanced lifestyle counseling and dieting program (the diet group, or DG) or to enhanced lifestyle counseling, dieting, and a supervised physical training program (the diet and training group, or DTG). Individuals diagnosed with MetS were analyzed in this study (DG: N = 09; DTG: N = 14). Before and after the intervention, the participants' anthropometric measures and cardiovascular disease (CVD) risk factors were assessed. Following the interventions, significant improvements were noted in all anthropometric variables among all participants (p ≤ 0.001 for all). Significant differences were noted between groups in terms of body weight (p ≤ 0.05), waist circumference (WC; p ≤ 0.01), body mass index (BMI; p ≤ 0.05), fat percentage (F%; p ≤ 0.05), and waist-to-hip ratio (WHR; p ≤ 0.05). All MetS components also improved in both groups, and the most significant improvements were observed among the training group in terms of fasting blood glucose (FBG) level (p ≤ 0.05), triglyceride (TG) level (p ≤ 0.001), overall cholesterol (OC; p ≤ 0.01), low-density lipoprotein cholesterol (LDL-c; p ≤ 0.05), and very low-density lipoprotein cholesterol (VLDL-c; p ≤ 0.001).
Conclusion: A minor daily caloric restriction (of approximately 500 kcal) program could be an effective tool in combating MetS. Further, the introduction of three weekly aerobic and resistance-training sessions in a gymnasium to the caloric restriction program may deliver better outcomes, mainly in terms of reductions in body weight, WC, FBG level, TG level, OC, LDL-c, and VLDL-c.


[GUIDELINES]
No

[STEPS]
1.
TITLE: E. coli Culture
AUTHORS: Bailey  Clark

[DESCRIPTION]
The abstract will be added later.

[BEFORE_START]
A video if unfamiliar: https://youtu.be/TWt62pgAEh0

[STEPS]
SECTION: Medium and Antibiotic Preparation
1. Add 2 mL (3 mL  also acceptable) of LB medium and the appropriate antibiotic that selects for the target strain into a 15 mL tube. 
 


SECTION: Medium and Antibiotic Preparation
2. Place the 15mL tube in the ThermoMixer at 37°C, 300 rpm, for 5 min . 
 


SECTION: Retrieving Cells
3. Gather cells. 
SECTION: Retrieving Cells
3.1. Swab a single colony on the agar plate with a sterile 1µL inoculation loop.
   

SECTION: Retrieving Cells
3.2.  OR Dip a sterile 10 µL innoculation loop in a preexisting culture tube. 

SECTION: Innnoculation
4. Innoculate the 15 mL tube with the loop. 
SECTION: Innnoculation
5. Place the 15 mL tube in the thermomixer for 16 hours at 37°C and 300 rpm.
TITLE: Pancreatic Insulin Content by Acid-Ethanol Extraction
AUTHORS: Ed Leiter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Used to calculate the insulin content in the pancreas.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bime4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimf4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimg4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimh4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimi4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimk4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bimm4kcn.jpg" /></div></div>

[STEPS]
?. ¼ - ½ of the pancreas is placed into 5 ml Acid-Ethanol (1.5% HCl in 70% EtOH) in a 15 ml conical vial.
?. Incubate O/N at -20ºC.
?. Homogenize tissue (I use a Polytron homogenizer).
?. Incubate O/N at -20ºC.
?. Centrifuge at 2000 rpm 15 min at 4ºC (Sorvall RT6000).
?. Transfer aqueous solution to a new 15 ml conical vial.
?. Neutralize 100 µl of Acid-Ethanol extract with 100 µl 1M Tris pH 7.5.
?. Dilute further (1:100, 1:1000, or 1:5000 depending upon the strain) in Insulin ELISA sample diluent.
?. Run diluted sample on Insulin ELISA (Exocell). Calculate ng/ml with appropriate dilution factor.
?. Run 20 µl of the neutralized solution in a Bradford Assay (250 µl Coomassie Blue Reagent, Thermo Scientific) against a standard curve. Calculate µg/ml with appropriate dilution factor.
?. Divide Insulin content ng/ml by Protein content µg/ml.