eamag/protocols-with-steps huggingface dataset:
TITLE: Microscopy-based bead protein-protein interaction assay
AUTHORS: Elisabeth Holzer

[DESCRIPTION]
This protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.

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

Purify tagged bait and prey proteins
 


[GUIDELINES]
Experiment should be repeated at least three times for statistical analysis.

[STEPS]
SECTION: Prepare bait-coated beads
1. Equilibrate 20 µL Glutathione Sepharose 4B or RFP-Trap Agarose beads with 200 µL SEC buffer
 


SECTION: Prepare bait-coated beads
2. Incubate equilibrated beads with GST- or mCherry-tagged bait protein for a final concentration of 5 micromolar (µM) in SEC buffer for 60 min at 4 °C with gentle rotation.

SECTION: Prepare bait-coated beads
3. Centrifuge beads at 3000 rcf for 2 min at 4 °C

SECTION: Prepare bait-coated beads
4. Remove the supernatant and wash beads with 200 µL SEC buffer

SECTION: Prepare bait-coated beads
4.1. Repeat for a total of 2 washes, then discard buffer
SECTION: Prepare bait-coated beads
5. Add 20 µL SEC buffer to achieve a beads:buffer ratio of 1:1

SECTION: Interaction assay set-up
6. Pipette prey proteins into the wells of a 384-well glass-bottom microplate (Greiner Bio-One)
 

 

 

SECTION: Interaction assay set-up
7. Pipette 1 µL of bait-coated beads into each well

SECTION: Interaction assay set-up
8. Incubate the plate for 30 min in the dark at Room temperature 

SECTION: Signal detection
9. Use a microscope configured to detect fluorescent signal (e.g. Zeiss LSM 700 confocal microscope equipped with Plan-Apochromat 20X/0.8 objective)
SECTION: Signal detection
10. Acquire fluorescent images in the middle section of the beads and collect more than one image for each well
SECTION: Signal detection
11. Also acquire bright field images for each field
SECTION: Quantification using ImageJ
12. Draw several lines across each bead in the fluorescent channel and measure the intensity along the lines
SECTION: Quantification using ImageJ
13. Record the maximum intensity for each bead
SECTION: Quantification using ImageJ
14. For background correction, measure the average intensity of a rectangular ROI that covers an area of each field of view with no beads
SECTION: Quantification using ImageJ
15. Subtract the average intensity of the background ROI from each bead maximum in that field
SECTION: Quantification using ImageJ
16. Calculate the average of the background-corrected maximum intensities of beads for each sample
TITLE: PEI/Laminin Coating
AUTHORS:  Zoe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare plates for iPSC-derived neuron maturation. It is a part of the NGN2 cortical neuron differentiation protocol: coat plates in preparation for Day 7 dissociation and replating.</div></div>

[STEPS]
?. [PEI Coating]
At least one day before plating, make 2x borate buffer with  20x borate buffer (Thermo, cat. no. 28341) in  water in a  Falcon tube.
4 mL
36 mL
50 mL
?. [PEI Coating]
Dilute  5% PEI (poly-ethylenimine; stored at ) in  2x borate buffer to make 0.1% PEI.
800 µl
4 °C
40 mL
?. [PEI Coating]
Filter-sterilize with SteriFlip: attach filter and tube to  tube with PEI solution; flip and attach to vacuum; when medium drains through, remove filter and original tube.
50 mL
?. [PEI Coating]
Add  to each of the inner 60 wells of a 96 square-well plate (Brooks, cat. no. MGB096-1-2-LG-L);  for wells of 24-well plate (Corning, #3527).
150 µl
500 µl
Note: Poly-ornithine-laminin pre-coated plates (6-well, 24-well) can be substituted for this plating protocol-only 96-well must be PEI/laminin coated.
?. [PEI Coating]
On 96-well plates, leave a border of empty wells to avoid edge effects; fill these with PBS to maintain humidity.
?. [PEI Coating]
Label plate(s), wrap sides with parafilm and cover in plastic wrap; store at .
4 °C
?. [Laminin Coating]
On the day of passaging, make laminin  in  PBS (with calcium and magnesium ions), ex.  PBS with  laminin (Sigma, cat. no. L2020, ).
4 °C
1 mL
5 µl
?. [Laminin Coating]
Wash PEI-coated plates twice with distilled water ( for 96-well plates).
300 µl
?. [Laminin Coating]
Wash once with PBS.
?. [Laminin Coating]
Add  laminin to wells of 96-well plate, and  to wells of 12-well.
150 µl
0.5 mL
?. [Laminin Coating]
Incubate plates at least 2 hours at  before re-plating neurons.
37 °C
TITLE: Euplotes crassus micronuclear enrichment by PFGE
AUTHORS: Rachele Cesaroni, Angela Piersanti

[STEPS]
?. Starved Euplotes crassus cells (treated overnight with ampicillin 100 mg/ml) were filtered and harvested by centrifugation at 400 rcf for 3 min.
?. As much as possible of the sea water was removed and cells were resuspended in 1V of 1XTE buffer and mixed with  1V of 2% Certified Low-Melt Agarose (Bio-Rad) in 1XTE buffer, then quickly transferred into the wells of the plug mold to produce the plugs (around 150,000 cells in 80 ul of volume in each well).
?. Plugs were set at 4°C for at least 30 min and then placed in 5 ml cells lysis buffer (1% N-Lauroylsarcosine sodium salt, 1% SDS, 1 mg/ml Proteinase K, 0.5 M EDTA pH 8.0) at 60°C overnight.
?. The plugs were washed three times in 0.5 M EDTA pH 8.0 for 10 min each, then inserted into the wells of the 1% Certified Low-Melt Agarose gel (Bio-Rad).
?. The instrument used for the PFGE was CHEF Mapper XA Pulsed Field Electrophoresis System (Bio-Rad). 2.2 L of 0.5X TBE buffer were poured into the electrophoresis chamber for a pre-run of 30 min at 14°C, with a pump speed setting of 70 (flow rate of ~ 1 liter/minute).
?. The run was set with a gradient of 6 V/cm, a time of 30 h, an included angle of 120°, an initial switch time of 70 sec, a final switch time of 70 sec and a linear ramping factor.
?. When the run was completed the gel was stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific) for 60 min and visualized with blue light.
?. The bands of interest were cut and melted at 65°C for around 30 min. The gel volume was determined and digested with Agarase from Pseudomonas atlantica (Sigma) at 40°C overnight and then with RNase A (Thermo Fisher Scientific) 10 µg/ml for 2 h at 37°C.
?. . The DNA was extracted by Phenol/Chloroform, precipitated by 3 M sodium acetate pH 5.2/absolute EtOH, resuspended in 30 ml of ddH2O, and quantified by NanoDropTM 1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific).
TITLE: Thawing primary leukemia cells
AUTHORS: Kathrin  Bernt

[DESCRIPTION]
This protocol is used to thaw primary cells. Key points are the addition of DNAse, which helps to preserve viability.

[STEPS]
1. Transfer the contents to a 15ml conical tube.
2. Add DNase. 1/10 of the volume of liquid in the tube (100µl/1ml of sample). Do not pipette. Gently shake the sample to mix in the DNase.
3. Incubate sample in 37˚C water bath for 90 seconds.
4. Add media. 10ml, Drop for drop or very slowly while again gently mixing the sample.
5. Spin. @1600rpm for 6-7minutes.
6. Aspirate media.
7. Add DNase, 70µl/ 1ml of sample.
14. Count cells.
8. Resuspend cells in DNase by flicking the pellet.
9. Add media, 10ml, again very slowly, then resuspend cells after adding about half of the media by pipetting very gently up and down (or inverting the tube once all the media is added)
10. Spin @ 1600rpm for 6-7 minutes.
11. Aspirate media.
12. Add DNase. 50µl per 1ml of sample.
13. Resuspend in desired media. 
15.  Note: •	The use of DNase is especially important if you use PBS. It will prevent a lot of clumping. Adding the medium very slowly and continous resuspending of the cells will also greatly reduce clumping.
TITLE: Gcase co-immunoprecipitation
AUTHORS: michela.deleidi, Federico   Bertoli

[DESCRIPTION]
We developed this protocol to identify protein-protein interactions between the enzyme glucocerebrosidase (GCase) and other proteins in human iPSC-derived Neural Precursor Cells.

[STEPS]
SECTION: Gcase co-immunoprecipitation
1. Wash cells 1X with phosphate-buffered saline (PBS, Sigma‒Aldrich) and detach using Accutase.
SECTION: Gcase co-immunoprecipitation
2. Pellet the cell suspension at 280 rcf, 5 min, 23 °C.
SECTION: Gcase co-immunoprecipitation
3. Lyse the pellets in IP/lysis buffer (Thermo Fisher, #87787) supplemented with a  protease/phosphatase inhibitor cocktail (Pierce, #A32959). 
SECTION: Gcase co-immunoprecipitation
4. Carry out coimmunoprecipitation using the Thermo Fisher Pierce Crosslink Magnetic IP/Co-IP Kit (#88805) according to the manufacturer´s instructions:
SECTION: Gcase co-immunoprecipitation
5. Prewash 25 µL of  Pierce protein A/G magnetic beads (Thermo Fisher, #88802-3) twice with 1X Modified Coupling Buffer and incubate with 10 µg of GBA MaxPab polyclonal rabbit antibody (Abnova) or normal rabbit IgG (Covalab, #pab01004-P) on a rotating wheel   at4 °C. 
SECTION: Gcase co-immunoprecipitation
6. The following day, crosslink the antibody to the beads with a 0.25 millimolar (mM) DSS solution for 60 min on a rotating wheel at Room temperature.
SECTION: Gcase co-immunoprecipitation
7. Incubate crosslinked magnetic beads 60 min with a total of 7 mg of protein for each lysate. 
SECTION: Gcase co-immunoprecipitation
8. Elute Coimmunoprecipitated proteins from the beads with 60 µL of the kit-provided Elution buffer pH 2.0 (Pierce, #88805) and neutralize with 6 µL of Neutralization Buffer provided with the kit. 
SECTION: Gcase co-immunoprecipitation
9. Prepare samples for Western blotting by adding 5x Lane buffer +10% DTT 1 Molarity (M) to a final concentration of 1X.
TITLE: Aortic Banding in Mice
AUTHORS: Dale Abel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes the procedure used by the DiaComp for inducing pressure overload hypertrophy in mice.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bh3b4kcn.jpg" /></div></div>

[STEPS]
?. Mice are anesthetized with 1% isoflurane delivered via nose cone. A topical depilatory agent is applied to the neck and chest and the area is cleaned with betadine and alcohol. Mice are placed supine and temperature maintained at 37ºC with a heating pad. A horizontal skin incision ~ 0.5 - 1.0 cm in length is made at the level of the suprasternal notch. The thyroid is retracted and a 2 - 3-mm longitudinal cut is made in the proximal portion of the sternum. This allows for visualization of the aortic arch under low power magnification. A wire with a snare on the end is passed under the aorta between the origin of the right innominate and the left common carotid arteries. A 6-0 silk suture is snared with the wire and pulled back around the aorta. A bent 27-gauge needle is then placed next to the aortic arch and the suture is snugly tied around the needle and the aorta. Following ligation, the needle is quickly removed. The skin is closed and mice are allowed to recover on a warming pad until they were fully awake. The sham procedure is identical except that the aorta is not ligated.
TITLE: Plasmid used for transfection trails of Hematodinium 
AUTHORS: Imen  Lassadi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Attempts to transfect</span><span style = "font-style:italic;"> Hematodinium </span><span>was performed using a plasmid that will express eGFP under the control of DVNP regulatory sequences. See attachment for plasmid information. </span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.</p>

[STEPS]
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TITLE: Case - Intraperitoneal Glucose Tolerance Test
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">This is the standard protocol for most routine glucose tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or 6 hours.</div></div>

[STEPS]
?. Fast mice overnight or for 6 hours. Remove mouse from cage and put into a clean cage with water and no food (5:00PM). Next day begin GTT by 9:00AM.
?. Weigh each mouse and record weight.
?. Insert glucose strip into glucometer and check that the code matches for the strip being used.
?. Take fasting blood glucose by snipping the tail and putting a drop on the glucose strip (already in glucometer). Record fasting glucose as time 0’. Dip tail into clotisol to clot blood.
?. Calculate the amount of glucose needed for a concentration of 2gm glucose/kg body weight. Record the volume for each mouse.
?. Inject each mouse 3’ apart for time 0. Repeat #4 at 15min, 30 min, 60 min and 120 min after IP injection of glucose.
?. Do not take extra blood for insulin measurements. This causes too much stress. Use a separate group of mice for collecting blood after glucose injection for insulin measurements.
TITLE: Testing repetitive tDCS on daily smoking behaviour: A placebo controlled EMA study
AUTHORS: Ilse Verveer, Danielle Remmerswaal, Joran Jongerling, Frederik M. van der Veen, Ingmar H. A. Franken

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The aim of the study was to explore the effects of repetitive tDCS on smoking behaviour by means of ecological momentary assessments (EMA) in a sample of </span><span style = "font-style:italic;">ad libitum </span><span>smokers</span></div><div class = "text-block">Participants recruited for this study were smokers between the age of 18 and 65 years who smoked 10 cigarettes or more a day and who had the ability to speak, read, and write in Dutch. Exclusion criteria were: 1) Current substance use disorder of a substance other than nicotine or caffeine; 2) History of neurological or psychiatric disorders; 3) Any contraindication for electrical brain stimulation procedures (i.e. electronic implants or metal implants); 4) Pregnancy or breast-feeding; 5) Intentions to actively try to quit smoking in the next three months. Participants were recruited via advertisement at Erasmus University Rotterdam from October 2016 until March 2018 and received either course credit or a financial compensation of 20 euro</div><div class = "text-block">The current study employed a double-blind, randomized, sham-controlled design in which subjects received a total of six tDCS sessions (active or sham) on three days in one week with at least one day in between. Participants were first randomly assigned to either sham or active tDCS. Then, before the tDCS sessions and at three months follow-up, participants completed the Fagerström Test of Nicotine Dependence (FTND; [28]). Breath carbon monoxide concentrations were also measured using a Micro+ Smokerlyzer (Bedfont Scientific Ltd., Rochester, UK) to objectively define smoking.</div><div class = "text-block">To measure changes in smoking behaviour, participants were asked to keep track of their cigarette consumption, craving and affect in an application on their mobile phones (EMA). Questions in the application were presented at four random times a day for three weeks in total (random assessments; RA’s), starting the week before the first tDCS session. After three months, participants were asked to fill out the same four-time daily random assessments for one more week. In addition, participants were asked to start a session every time they smoked a cigarette (user-initiated smoking assessment; SA) for three months in total. An ‘end of the day’ assessment (EA) was also implemented for three months, which asked participants to fill out the total number of smoked cigarettes of that day. </div></div>

[STEPS]
TITLE: TS Procure 812 - primary fixation formalin/wet tissue (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
The method is used to process formalin fixed histological tissues to Procure 812 resin blocks. 

[GUIDELINES]
Sample is not optimal for EM, best to dissect and use only the outer 2mm of tissue mass. Sample is usually derived from histology.

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR & STEPS:




OPERATOR & STEPS:
SECTION: Primary fixation
2. Formalin for undefined extended period of time at Room temperature 

SECTION: Conventional
3. 2.5 % volume   in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 40 min  or overnight

SECTION: Conventional
4. 0.1 Molarity (M) buffer 15 min 

SECTION: Conventional
5. 1 % volume   in buffer 60 min 

SECTION: Conventional
6. 70 % volume   for at least 20 min  or overnight

SECTION: Conventional
7. 95 % volume    20 min 

SECTION: Conventional
8. 100 % volume    20 min 

SECTION: Conventional
9. 100 % volume    dried with molecular sieve 20 min 


SECTION: Conventional
10. 50 % volume   50 % volume   20 min 

SECTION: Conventional
11. 100 % volume   20 min 

SECTION: Conventional
12. 50 % volume   50 % volume Procure 812


SECTION: Conventional
13. 100 % volume Procure 81220 min 

SECTION: Conventional
14. 100 % volume Procure 812 60 min 

SECTION: Conventional
15. Embed & polymerise overnight in oven at 60 °C
TITLE: DNA Fragmentation &amp; Library Construction
AUTHORS: Marina McCowin

[DESCRIPTION]
The KAPA HyperPlus Kit provides a versatile, streamlined DNA fragmentation and library construction protocol for the rapid preparation of libraries for Illumina sequencing. The one-tube chemistry and protocol improves the efficiency and consistency of library construction, and yields libraries of similar or better quality than those produced with the KAPA HyperPrep Kit from Covaris sheared DNA. It outperforms tagmentation-based workflows in terms of robustness, flexibility and sequence coverage and uniformity.

[BEFORE_START]
Shipping and Storage
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and
storage. KAPA HyperPlus Kits are shipped on dry ice or ice packs, depending upon country of destination. Upon
receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When
stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date
indicated on the kit label.

Handling
Always ensure that KAPA HyperPlus Kit components have been fully thawed and thoroughly mixed before use.
The End Repair & A-Tailing Buffer and Ligation Buffer may contain precipitates when thawed at 2°C to 8°C. These
buffers must be thawed at room temperature and vortexed thoroughly before use. KAPA HiFi HotStart ReadyMix (2X)
contains isostabilizers and may not freeze completely, even when stored at -15°C to -25°C. Nevertheless, always
ensure that the ReadyMix is fully thawed and thoroughly mixed before use. Reaction master mixes prepared from
the enzymes and buffers for fragmentation, end repair and A-tailing, as well as for ligation, are very viscous and
require special attention during pipetting. Keep all enzyme components and master mixes on ice as long as possible
during handling and preparation.

Quality Control
All kit components are subjected to stringent functional quality control, are free of detectable contaminating exoand
endonuclease activities, and meet strict requirements with respect to DNA contamination.

[GUIDELINES]
The workflow combines enzymatic steps and employs minimal bead-based cleanups, thereby reducing sample
handling and overall library preparation time to 1.5 – 3 hrs. The kit contains all of the enzymes and reaction buffers required for:
1. enzymatic fragmentation to produce dsDNA fragments;
2. end repair and A-tailing to produce end-repaired,
5'-phosphorylated, 3'-dA-tailed dsDNA fragments;
3. adapter ligation, during which dsDNA adapters with 3'-dTMP overhangs are ligated to 3'-dA-tailed molecules; and
4. library amplification (optional), which employs highfidelity, low-bias PCR to amplify library fragments
carrying appropriate adapter sequences on both ends. 

[STEPS]
TITLE: Chemical fixation of Solarion arianae for transmission electron microscopy 
AUTHORS: Marek Valt, Pavla Hrubá

[DESCRIPTION]
This is an optimized version of the protocol for standard chemical fixation for transmission electron microscopy that was used for the fixation of the culture of Solarion arianae.

[STEPS]
SECTION: Cell harvest
1. Centrifuge a well-grown culture at 1250 g at 4 °C.
SECTION: Chemical cell fixation
2. Immerse pellet with 2,5% glutaraldehyde fixative in 0,1M cacodylate buffer solution for 1h. Work on ice.
SECTION: Cell harvest
1.1. Discard supernatant and collect pellet.
SECTION: Chemical cell fixation
3. Wash with 0,1M cacodylate buffer, 3 times.
SECTION: Chemical cell fixation
4. Post-fix with 2% OsO4 in 0,1 M cacodylate buffer for 1h, on ice
SECTION: Chemical cell fixation
5. Wash fixed samples in distilled water, 3 times.
SECTION: Chemical cell fixation
6. Dehydrate samples in ethanol series 30%, 50%, 70%, 80%, 90%, 95%, (100% 3 times)
SECTION: Chemical cell fixation
7. Impregnate the dehydrated sample with acetone 1:1 ethanol solution.
SECTION: Chemical cell fixation
9. Impregnate acetone 1:1 resin (EMbed 812) mixture.
SECTION: Chemical cell fixation
8. Impregnate with 100% acetone.
SECTION: Chemical cell fixation
10. Transfer the sample to (EMbed 812) resin. Repeat 3 times every 4h.
(can be left longer or overnight)

SECTION: Chemical cell fixation
11. Polymerize samples embedded in resin at 70 °C for 48h.
SECTION: Section preparation
12. Cut sections. 
For Solarion, 80nm thick sections were cut with a diamond knife on an Ultracut E ultramicrotome (Reichert).
SECTION: Section preparation
13. post-contrast with uranyl acetate and lead citrate.