eamag/protocols-with-steps huggingface dataset:
TITLE: Fluorescence measurement
AUTHORS: iGEM Dusseldorf

[STEPS]
?. Prepare 5 ml LB medium with antibiotic resistance and 0.5% ml Tergitol
?. Inoculate one colony in the prepared LB medium
?. Add 0.01-1 mM of the desired fatty acid to the culture medium
?. Inoculate the culture for at least 16 hours at 37 °C
?. Measure the absorption with a photometer at a wavelength 600 nm
?. Dilute the culture to an OD600 of 0.05 with LB medium
?. Fill a 24 well plate with 800 µl of 3 biological with 3 technically replicats respectively like shown in the table below
?. ABCDEF1Sample 1.1Sample 1.2Sample 1.3Sample 5.1Sample 5.2Sample 5.32Sample 2.1Sample 2.2Sample 2.3Sample 6.1Sample 6.2Sample 6.33Sample 3.1Sample 3.2Sample 3.3Sample 7.1Sample 7.2Sample 7.34Sample 4.1Sample 4.2Sample 4.3Sample 8.1Sample 8.2Sample 8.3
ABCDEF1Sample 1.1Sample 1.2Sample 1.3Sample 5.1Sample 5.2Sample 5.32Sample 2.1Sample 2.2Sample 2.3Sample 6.1Sample 6.2Sample 6.33Sample 3.1Sample 3.2Sample 3.3Sample 7.1Sample 7.2Sample 7.34Sample 4.1Sample 4.2Sample 4.3Sample 8.1Sample 8.2Sample 8.3
?. Inoculate the 24 well plate at 37°C for at least 16 hours
?. Transfer the samples from the 24 well plate on a 96 well plate like shown in the table below
?. ABCDEFGHIJKL1Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.32Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.33Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.34BlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlank5Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.36Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.37Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.38BlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlank
ABCDEFGHIJKL1Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.32Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.33Sample 1.1Sample 1.2Sample 1.3Sample 2.1Sample 2.2Sample 2.3Sample 3.1Sample 3.2Sample 3.3Sample 4.1Sample 4.2Sample 4.34BlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlank5Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.36Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.37Sample 5.1Sample 5.2Sample 5.3Sample 6.1Sample 6.2Sample 6.3Sample 7.1Sample 7.2Sample 7.3Sample 8.1Sample 8.2Sample 8.38BlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlankBlank
?. Measure the desired fluorescence at the appropriate wavelenght and the OD600 with a plate reader
TITLE: RNA Isolation from Plant Tissue Protocol 16: CTAB-Hot Acid Phenol Method for Algae
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Falicia Goh and Neil Clarke</div><div class = "text-block"><span>This RNA isolation method is a combination and modification of the hot acid phenol method (protocol 14) and that described by Asif et al</span><span style = "vertical-align:super;">6</span><span>. This method was used for two taxa (P. cruentum and B. braunii). </span></div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div><div class = "text-block"><span style = "vertical-align:super;">6</span><span> Asif, M.H., Dhawan, P. & Nath, P. A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Molecular Biology Reporter 18, 109‐115 (2000).</span></div></div>

[STEPS]
?. Preheat phenol and phenol:chloroform to .
65 °C
Heated phenol should not be re-used.
?. Collect algae cells via centrifugation for  at  at .
0 Room temperature
?. Re-suspend the frozen pellet in  of preheated extraction buffer.
800 µl
?. Incubate at  for . Gently vortex every .
65 °C
?. Cool to .
0 Room temperature
?. Collect the aqueous phase by centrifuging for  in micro-centrifuge at  at .
0 Room temperature
?. Collect aqueous phase and re-extract with an equal volume of chloroform.
?. Collect aqueous phase and add  LiCl to a final concentration of .
?. Recover the RNA by centrifugation at  at  for .
4 °C
?. Dissolve pellet in DEPC treated water.
?. Extract the aqueous phase with equal volume of phenol:chloroform (5:1).
?. Vortex for  at .
0 Room temperature
?. Extract the aqueous phase with equal volume of chloroform.
?. Collect aqueous phase and add 1/30 volume of  sodium acetate pH 5.5 and 0.1 volume of 100 % ethanol.
?. To the clear supernatant add  sodium acetate pH 5.2 to a final concentration of  and 3 volumes of 100 % ethanol.
?. Spin in micro-centrifuge at  at top speed for .
4 °C
?. Wash the pellet with 70 % ethanol.
?. Invert tubes and air dry at room temperature.
?. Resuspend pellets in  of DEPC treated water.
50 µl
?. Flash freeze pellets with liquid nitrogen and keep at   until extractions are carried out.
-80 °C
?. Add equal volume of chloroform.
?. Shake vigorously until 2 phases form an emulsion.
?. Centrifuge as above.
?. Allow the RNA to precipitate at  overnight.
4 °C
?. Spin for  in a micro-centrifuge at top speed.
?. Mix well and keep on ice for .
?. Centrifuge in cold for .
?. A white jelly-like pellet consisting mostly of polysaccharides is obtained and discarded.
?. Allow the RNA to precipitate at  for  to overnight.
-80 °C
?. Extract once with hot acid phenol.
TITLE: Dental emergency: scoping review protocol
AUTHORS: Karla Frichembruder, Camila Mello dos Santos, Fernando Neves Hugo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Introduction: </span><span>considering the high prevalence of dental caries, understand the factors that may influence the use of network health services in dental emergency cases is important to improve the access and qualify the emergency net care (ENC). To provide an overview about this field in Brazil we consider performing a scoping review. The aim of this publication is to present the protocol of a scoping review about dental emergency in public health services in Brazil.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Methods and analysis</span><span>: the review will follow the Joanna Briggs Institute scoping review methods protocol. We will search for relevant studies in MEDLINE, EMBASE, SCIELO and perform a gray literature search using Google Scholar. Two independent reviewers will select the articles. Discrepancies will be solved through discussion and, in case of disagreement, with a third reviewer. Descriptive statistical analysis will be performed.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Ethics and dissemination: </span><span>a paper summarizing the findings from this review will be published in a peer-reviewed journal. Ethical approval is not required. </span></div></div></div>

[STEPS]
TITLE: test_protocol
AUTHORS: isis.scott

[DESCRIPTION]
abstract 

[STEPS]
SECTION: Scope
1. write about the protocol and the reason to run this experiment
SECTION: Safety
2. Safety googles

SECTION: Safety
3. Lab coat 
SECTION: Safety
3.1. fire-resistant 
SECTION: Procedures
4. Add 10 mL of 1 1 Molarity (m) NaCl solution to soil at25 °C    in the oven
TITLE: one-step growth curve
AUTHORS: Jiaxin Li

[STEPS]
?. Subculture host bacterium in medium of choice plus 2 mM CaCl2 and grow to mid-log phase (ca. 0.5 OD650nm).
?. Pipette 9.9 mL of the log phage culture into the empty flask and place at the appropriate incubation temperature for 5 min.
?. Add 0.1 mL of phage preparation to the 9.9 mL culture. (FLASK A)
?. Transfer 1.0 mL from FLASK A to 9.0 mL of prewarmed medium, mix well. (FLASK B)
?. Transfer 1.0 mL from FLASK B to 9.0 mL of prewarmed medium, mix well. (FLASK C)
?. Place FLASK (A, B or C) at 37°C, 180 rpm
?. Every 10 minutes remove 0.1 mL from the appropriate FLASK (A, B or C) add to the molten OVERLAY; add 0.1 mL of PLATING HOST; mix and pour on surface of UNDERLAY plates. Each experiment should perform in triplicate.
?. When the overlays have hardened (ca. 15 min) invert the plates and place them in an incubator.
?. After an appropriate incubation period (ON for E. coli or Pseudomonas aeruginosa) count the plaques on each of the plates.
TITLE: Basic immunofluorescence protocol for adherent cells
AUTHORS: Girija Goyal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Adherent cells were cultured in flat bottom plates and fixed in situ. This is a protocol to detect specific proteins by immunofluorescence in these cells. Immunofluoroscence is a common laboratory technique where specific proteins within cells can be detected using antibodies coupled to fluorophores and then visualized using a microscope. The cells were counterstained with Hoechst dye to label nuclei.</div></div>

[STEPS]
?. [Fixation]
Fix the cells using 4% paraformaldehyde (PFA) by removing media and submerging cells in PFA for  at . Cells can be left in fixative for a few days at
[room temp]
[Fridge]
?. [Permeabilization]
For the antibody to be able to penetrate the cells, the membranes need to be permeabilized. This may be necessary even if the protein to be detected is expressed on the surface if the antibody binds an intracellular region. Aspirate PBS from previous step and replace with  permeabilization buffer and incubate forStore staining buffer in  fridge
100 µl
[Room temp]
?. [Fixation]
Aspirate fixative and replace with PBS. Cells can be stored at this step for upto 1 month.
?. [Permeabilization]
Aspirate permeabilization buffer and replace with  PBS. Aspirate PBS and proceed to next step or store in the fridge in fresh PBS.
100 µl
?. [Primary antibody staining]
Incubate in primary antibody (the antibody that detects the protein of interest) at the recommended dilution (1:100) in staining buffer . See guidelines for dilutions of specific antibodies and duration of incubation. Store staining buffer in  fridge
[Overnight]
?. If you plan to reuse the diluted antibody for another experiment, use a pipette to transfer it into a collection tube for storage. Otherwise, aspirate primary antibody from the cells, and add PBS and leave plate on shaker for at room temp. Repeat twice.
?. [Secondary staining]
For greater flexibility, primary antibodies are left unconjugated and detected using a secondary antibody which is specific to primary antibody (see guidelines) and are labeled with a fluorophore. Incubate in secondary antibody at the recommended dilution in staining buffer. See guidelines for dilutions of specific antibodies and duration of incubation.
?. [Post-secondary washing and counterstaining for nuclei/DNA]
Aspirate secondary antibody from the cells, and add PBS and leave plate on shaker for at room temp.
?. [Post-secondary washing and counterstaining for nuclei/DNA]
Replace PBS with 1:2000 dilution of Hoechst dye and incubate at room temp for
?. [Post-secondary washing and counterstaining for nuclei/DNA]
Conduct two washes with PBS as in step 8. The cells are ready for visualization on the microscope.
TITLE: Preparation of frozen nuclei for single-nucleus RNA seq
AUTHORS: Kimberly Siletti, Sten Linnarsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes our preparation of nuclei for single-nucleus RNA sequencing on the 10X Genomics Chromium system, starting from FAC-sorted frozen nuclei. </div></div>

[STEPS]
TITLE: Dissection and immunohistochemistry of mouse brainstem
AUTHORS: Seol-Hee Kim, Thomas Taylor-Clark

[DESCRIPTION]
Mice are euthanized, perfused with fixative for brainstem tissue collection. Mouse brainstem is cryosectioned and slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins are visualized using microscopy.

[STEPS]
SECTION: Tissue collection and cryosection
1. Mice are euthanized by CO2inhalation and transcardially perfused with ice-cold PBS followed by perfusion fixation with ice-cold 3.7% formaldehyde (FA).
SECTION: Tissue collection and cryosection
2. Brainstem is dissected out and post-fixed for 4 h in 3.7% FA at 4°C. The brainstem is washed in PBS to remove residual FA and transferred to 30% sucrose solution for cryoprotection.
SECTION: Tissue collection and cryosection
3. The brainstem is mounted in OCT (optimal cutting temperature) compound and snap frozen in dry ice. The brainstem is sectioned in 40 μm slices.

SECTION: Tissue collection and cryosection
4. All slices are collected onto SuperFrost Plus slides. Slides are then air dried at room temperature in the dark overnight.
6. Tissue are blocked with 1% bovine serum albumin (BSA)/10% donkey serum (DS)/0.3% PBSTx. for 1 h at room temperature.

SECTION: Immunohistochemistry
5. Tissue are permeabilized with 0.3% Triton X-100 in PBS (PBSTx) for 15 min.
7. Tissue are incubated with primary antibodies diluted in blocking buffer overnight at 4°C.
8. Tissue are washed with 0.2% Tween 20 in PBS (PBST) three times for 10 min. 
9. Tissue are incubated with secondary antibodies in 1% BSA/5% DS in 0.2% PBST for 1 h at room temperature.
10. Tissue are washed with 0.2% PBST three times for 10 min and rinsed briefly with H2O. 
11. Slides are air-dried and mounted with DPX Mounting Medium (Sigma-Aldrich) for imaging.
TITLE: Peptide N-Terminal Modification
AUTHORS: Cathy  Miller

[DESCRIPTION]
Although many of the most widely recognized post-translational modifications are characteristic of secretory or cell-surface proteins, most proteins, whatever their ultimate cellular desination, undergo some modification. For proteins synthesized completely within the cytoplasm, the earliest and most widespread are removal or modification of the N-terminal residue. In many proteins the N-terminal α-ammonium group (PK=8) undergoes secondary modification.


N-terminal modification reduces overall solubility of the peptide by reducing its overall charges. However, the stability of the peptide could also be increased because N-terminal modification generates a closer mimic of the native protein. Therefore, these modifications might increase the biological activity of a peptide and prevent degradation by enzymes.

Creative Peptides is specialized in the custom synthesis of N-terminal modification peptides, providing a confidential and efficient service at competitive prices. Every step of peptide synthesis is subject to Creative Peptides' stringent quality control.
Typical delivery specifications include
HPLC chromatogram
Mass spec analysis
Synthesis report
Certificate of analyses

[STEPS]
TITLE: Intramuscular Injection Adult Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the general procedures used for intramuscular injection in adult mice.  </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Self-made chrome alum gelatin coated slides
AUTHORS: Sonja Fritzsche

[DESCRIPTION]
This is step-wise protocol to coating microscopy slides with chrom alum gelatin for better tissue adherence.

[STEPS]
SECTION: Prepare chrome alum gelatin solution
1. Heat up 200 mL ddH2O to 50 °C on hot plate using a magnetic stirrer.

SECTION: Prepare chrome alum gelatin solution
2. Add 1 g gelatin gradually until completely dissolved to prevent formation of clumps. Solution has to be clear.

SECTION: Prepare chrome alum gelatin solution
3. Let solution cool down to room temperature and add 0.1 g Chrome potassium sulfate until completely dissolved.
Either proceed immediately or store the solution for a few days @ 4 °C. If stored, let the solution warm-up to room temperature
before use.

SECTION: Slide coating
4. Pour prepared chrome alum gelatin solution in a staining dish preheated at 60 °C.

SECTION: Slide coating
5. Incubate to-be-treated slides in 70 % (v/v) Ethanol for 10 min to clean them.

SECTION: Slide coating
6. Rinse in distilled water, dip at least 3 times to remove alcohol.
SECTION: Slide coating
7. Dip the slides 5 times in the solution, 10 s each.

SECTION: Slide coating
8. Leave the slides to drain for a few minutes onto a filter paper.
SECTION: Slide coating
9. Dry the treated slides in an oven at 37 °C over night.

SECTION: Slide coating
10. Store the slides in a dry box and protect from dust.
TITLE: Isolation of Nuclei from Adult_Human_Brain_Tissue
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis. 
 
Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Live-cell imaging; Calcium imaging with Fura-2
AUTHORS: Minee-Liane Choi

[DESCRIPTION]
This protocol describes a method to perform calcium imaging with Fura-2 (live-cell imaging)

[STEPS]
1. Cells are washed with HBSS (Cat No. 14025092, ThermofisherScientific)
2. Cells are loaded with 5uM of Fura 2-AM  (F1221, ThermofisherScientific) in HBSS. 
 

3. CCD protocol is set up by selecting a501 nm wheel (both 340 nm and 380 nm wavelength are excited b 501 nm).
TITLE: LB Agar
AUTHORS: sgoodwin

[DESCRIPTION]
Protocol for producing LB agar growth medium

[STEPS]
SECTION: LB Agar
1. Add 1.5 g LB powder, 1.5 g agarose powder to conical flask

SECTION: LB Agar
2. 
Dilute with 200 mL distilled water. Ensure fully dissolved

SECTION: LB Agar
3. Seal conical flask with sponge bung and foil
SECTION: LB Agar
4. Sterilise growth medium by autoclaving
TITLE: Isolation of Nuclei from Brain Tissue Using Gradient Centrifugation for Myelin Depletion for 10x Genomics Platform
AUTHORS: Allen Institute

[DESCRIPTION]
Protocol is used for isolation of nuclei from frozen brain tissue or
thawed and microdissected brain tissue sections for RNA-seq analysis. 

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number 
UM1MH130981. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

[STEPS]
TITLE: Aptamer 2-step conjugation protocol (INSA-Lyon 2016)
AUTHORS: Jorge Fernández

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for bioconjugation of amino terminal modified aptamers with carboxyl surface modified latex beads. Adaptated from iGem team INSA-Lyon 2016.</div></div>

[STEPS]
?. [Latex Beads Preparation]
Dilute  480 µL of 2.5 % wt beads stock with 120µL of additional PBS buffer. Reaching a final volume of 500 µL.
?. [Latex Beads Preparation]
Centrifugue the tube at 14.000 rpm for 10minutes. Discard the supernatant and resuspend them in 600 µL of MES buffer. For resuspending beads repeated pipetting it's highly recommended, aspiring and blowing out in the eppendorf tube. It's crucial assuring perfect beads disperssion, if little agreggates are appreciated, try reducing centrifugation times or sonicating the beads for resuspension (5 minutes sonication at moderate power).
?. [Latex Beads Conjugation]
Add quickly 12 mg of EDC (13.7 µL) , and let it stand for 15 min at room temperature.
?. [Latex Beads Conjugation]
Centrifugate at 14.0000 rpm for 10 minutes, and resuspend in 2 mL of MES  buffer.  Repeat this step twice.! We have modified this step, since INSA-Lyon original protocol used PBS for washing and conjugation steps. o-isoacylurea intermediate groups are very inestable , reducing their half life with the pH. At pH higher than 7 they ussualy reacts regenerating carboxyl groups.
?. [Latex Beads Conjugation]
Add 120 µL of amino terminal modified  Aptamer (6 µM) to each tube, and incubate them for at least two hours.
?. [Latex Beads Conjugation]
Aliquot the suspension, adding 500 µL in  four different eppendorf tubes.
?. [Washing and storage]
Centrifugate the mixture at 14.000 rpm for 10 minutes  and wash  the pellet twice with PBS + Tween 0.01% w/v and BSA 0.1% w/v. The beads  can be stored a few weeks in this buffer. Do not freeze them.
TITLE: Determination of IgG concentration by the Mancini test.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. An appropriate anti-IgG antiserum (antibody) is poured in the center well of an agar-containing plate.
?. Carefully circular wells are cut and detached from the plates.
?. A series of standards containing known concentrations of IgG are placed in separate wells, while “unknown” human serum samples and control are placed in other wells.
?. A ring of precipitate forms in the area of optimal antigen-antibody concentration, meaning anti-IgG - IgG reaction as the antigen diffuses radially.
?. The diameters of the rings are measured and perceived normally in 48-72 hours.
?. Finally, a standard curve is developed using the ring diameters of the standards versus the concentrations.
?. A curve is then used to plot the concentration of the control and unknown IgG samples.
TITLE: Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases
AUTHORS: Peter Simons, Virginie Bondu, Angela Wandinger-Ness, Tione Buranda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-</span><span style = "font-style:italic;">S</span><span>-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.</span></div></div>

[STEPS]