eamag/protocols-with-steps huggingface dataset:
TITLE: Improving Diagnosis in Cognitive Disorders
AUTHORS: Laura McWhirter, Craig Ritchie, Alan Carson, Jon Stone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Functional cognitive disorders are likely to account for a significant proportion of adults presenting with cognitive (memory and thinking) symptoms which are not caused by neurodegenerative disease or structural brain lesions, incorporating those with isolated functional cognitive symptoms, those with primary psychiatric disorders, and those with health anxiety or who attend excessively to normal cognitive lapses. There is a need for improved diagnostic methods to identify those with functional cognitive symptoms so that they might be offered appropriate treatment, protected from iatrogenic harms, and also in order to improve the specificity of prodromal neurodegenerative disease phenotypes.</div><div class = "text-block">This study aims to identify elements of clinical, neurological, cognitive examination which can, in combination as part of a diagnostic scoring tool, accurately discriminate functional cognitive symptoms from those more likely to be due to neurodegenerative disease. The study will recruit 120 participants with mild or subjective memory symptoms (but not dementia) from memory, neurology, and neuropsychiatry clinics. Each subject will attend for a structured research assessment examining interactional, linguistic, metacognitive features, as well as psychiatric, physical, and sleep disorder comorbidity. Multiple outcomes will be compared with panel consensus diagnosis using discriminant factor analysis. The intention is to follow up medical record / general practice diagnosis at 1-2 years, and ultimately it is hoped that further studies beyond this will re-examine the cohort in the future. </div></div>

[STEPS]
TITLE: Collecting Spleens for genotyping rats
AUTHORS: Gabriel J  Barrero, Oksana  Polesskaya, Abraham  Palmer

[DESCRIPTION]
This protocol describes how to dissect and collect soft tissue (spleen) from rats. Collected spleen tissue is intended to be used for DNA extraction and genotyping

[STEPS]
SECTION: Materials
1. Rats are euthanized as per IACUC guidelines
Spray fur with ethanol. Pick up skin above the sternum, and make a cut through skin and muscle layers.
Don’t cut the chest cavity (don’t go above the diaphragm).  

SECTION: Materials
2.  Cut skin and muscles from the sternum down to the lower belly. Mostly you will see liver and colon. (Note: After some practice, you will be able to make the small precise cut on the left side of the belly to reach the spleen without extensive dissection)
     

SECTION: Materials
3. Lift liver lobes and push aside fat tissue and intestines. You will see dark-colored organs – kidney and spleen.
The kidney is bean-shaped. Spleen is elongated. Pull-on the spleen gently to remove from the specimen, trim off fat
  

SECTION: Materials
4. Put spleen into a pre-labeled 2 mLmicrocentrifuge tube (for example, Fisher Scientific cat#05-408-138). Add approximately 0.5 mL saline solution in the tube with the spleen to prevent dry freezing of tissue, but make sure that tube is not over-filled. If your rats have been implanted with an RFID chip, try to find this chip during dissection and put it directly into the tube with the spleen. We will read both the barcode and RFID chip to confirm sample identity. For labeling use “Cryo Babies” for laser printers (Diversified Biotech cat# LCRY-1700), they withstand long-term storage in -80 °C without peeling off the tubes.
    

SECTION: Materials
6. 
Store samples in cardboard boxes clearly labeled with the investigator's name, project, tissue, box number (if out of many), and date. Example: JHOU-HSratsU01-Spleen-Box02_25Feb2019
  

SECTION: Materials
5. 
Cut off  ~ 5 mm of the tail tip and store it as a backup for genotyping at your lab. Use 2 mLtube labeled with the barcode and rat ID, add saline to prevent sample desiccation. Store at-80 °C long-term

SECTION: Materials
7. Shipping spleens
SECTION: Materials
7.1. Use at least 10 kg (20 lb) dry ice for shipping. If you have a choice, use pellets. If you use slabs, make sure that there is no free space in the shipping box, so slabs cannot move freely and crash things.
·
SECTION: Materials
7.2.  For shipping use Styrofoam box that is surrounded by a cardboard box. Choose boxes with thick walls. For example, ULINE S-13392 or ULINE S-11357.

SECTION: Materials
7.3. · Send by FedEx overnight on Monday or Tuesday, check for holidays. It is best if you send an email to the lab beforehand so they are ready to receive it.

SECTION: Materials
7.4. Send a list of samples that you are shipping (RFIDs), we will use it to update the database.
TITLE: Crystallization of SARS-CoV-2 N Protein
AUTHORS: Peter Marples, Lizbé Koekemoer, Daren Fearon

[DESCRIPTION]
The crystallization protocol and buffer conditions used to obtain reproducible SARS C0V-2 Nucelocapsid  crystals suitable for XChem fragment screening.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
57.6 µL20 mg/mL  
2.88 mL 
5.76 µL   seeds, dilution 1:100

SECTION: Crystallization experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 200 nL20 mg/mL    to each lens using the SPT mosquito.
Dispense  180 nL to each lens using the SPT mosquito.
Dispense  20 nL to each lens using the SPT mosquito.

Drop ratio: 5:9:1 ratio (100 nL   : 180 nL reservoir solution: 20 nL seeds)
Final drop volume: 300 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
 0.1 Molarity (M) HEPES pH 7.8 
10 % isopropanol
23% w/v PEG 4000

Stock solutions used:
1 Molarity (M)  HEPES adjusted to pH 7.8  with NaOH
70 % isopropanol  
50% w/v PEG 4000

 

SECTION: Crystallization experiment
7. Crystal form after ~12 h.
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager. 

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock: 
 
1: 100 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.68
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: Multi-Contact 4C (MC-4C): long molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies
AUTHORS: Carlo Vermeulen, Amin Allahyar, Britta A.M. Bouwman, Peter H.L. Krijger, Marjon J.A.M. Verstegen, Geert Geeven, Christian Valdes-Quezada, Ivo Renkens, Roy Straver, Wigard P. Kloosterman, Jeroen de Ridder, Wouter de Laat

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We present the protocol and data analysis toolbox for Multi-Contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromosome conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. In contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long molecule sequencing on Oxford Nanopore or Pacific Biosciences platforms, thus allowing study of multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox employing data-intrinsic background models then discerns whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously happening at chromatin hubs. The entire procedure can be completed in two weeks and requires access to a third generation sequencing platform. 
 
 
 </div></div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: HuBMAP | GE HealthCare/UPitt Cell DIVE™ and scRNA Modality Overview
AUTHORS: Liz McDonough

[DESCRIPTION]
This is an overview of all protocols currently in use for the 2024 GE HealthCare/UPitt collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.

[STEPS]
SECTION: Obtaining Donors and Tissue Blocks
1. Confirm donor acceptance criteria for inclusion.

Donor Acceptance Criteria for GE/UPitt HuBMAP Inclusion
SECTION: Obtaining Donors and Tissue Blocks
2. 5 mm skin biopsies obtained from donor subjects are divided into two parts, with one half for formalin fixed and paraffin embedded (FFPE) processing and the other half for skin digestion for further scRNA analysis.
SECTION: Cell DIVE Multiplexed Immunofluorescence (MxIF)
4. Prepare formalin fixed and paraffin embedded (FFPE) blocks and tissue sections.

HuBMAP | Formalin Fixation and Paraffin Embedding of Tissue Samples
HuBMAP | Sectioning of FFPE Specimens
SECTION: Cell DIVE Multiplexed Immunofluorescence (MxIF)
6. Characterize antibodies (primary/secondary, direct conjugates, and zenon labelled antibodies) and determine any antigen effects from the Cell DIVE dye inactivation process.

Cell DIVE™ Platform | Antibody Characterization for Multiplexing 
Cell DIVE™ Platform | Antibody Staining & Imaging

SECTION: Cell DIVE Multiplexed Immunofluorescence (MxIF)
7. Prepare direct conjugates for study.

Cell DIVE™ Platform | Antibody Purification Chemistry
Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
SECTION: Cell DIVE Multiplexed Immunofluorescence (MxIF)
8. Perform Cell DIVE™ multiplexed data acquisition on the final cohort. 

 

SECTION: scRNA Analysis
3. Perform skin digestion and scRNA analysis.

HuBMAP | Digestion and scRNA Analysis of Skin
SECTION: Cell DIVE Multiplexed Immunofluorescence (MxIF)
5. Deparaffinize and rehydrate slides.

Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval
TITLE: Blood pressure
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Blood pressure measurement in mice

[STEPS]
1. Measure blood pressure using the tail-cuff method (LE-5002 Non-Invasive Blood Pressure Meter, Panlab).
2. Assess the systolic and diastolic blood pressure (in mmHg) in two independent sessions for each mouse.
3. Calculate the mean from the valid values of 10 measurements in each session.
TITLE: U Mass - Body composition (organs)
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure fat mass, lean mass, and water mass in individual organs. Fat, lean, and water mass composition in specific organs is altered in obesity.</div></div>

[STEPS]
?. Euthanize mice using intraperitoneal injection of pentobarbital.
?. Quickly dissect and extract specific organs to measure their composition.
?. Place specific organ into an instrument column.
?. Measure composition of organ using instrument standard operating procedure.
TITLE: Immunofluorescence staining of paraffin embedded cell block sections
AUTHORS: Erez Nissim Baruch, Rona Ortenberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is  designated for Immunofluorescence staining of paraffin embedded   cell block  sections. </div></div>

[STEPS]
?. After the slides were cut, let them dry in room temprature over-night, or 2-3 hours on a heating plate at 37°C.Before stainig incubate the slides in oven at 55°C for 1 - 2 hours
?. In a chemical hood:Wash in Xylene for 10 minutesWash in new Xylene for another 10 minutesWash in Ethanol 100% for 3 minutes
?. On the bench, room temprature:Wash in new Ethanol 100% for 3 minutes.Wash in Ethanol 95% for 3 minutes.Wash in Ethanol 70% for 3 minutes.Wash in 0.05% Tween-20 in PBS for 5 minutes.Wash again in new 0.05% Tween-20 in PBS for 5 minutes.
?. Microwave:1.Put the citrate buffer x 1 in a plastic container designated for microwave use.2.Put the slides in the container.3.Insert the container into the microwave and activate on maximal energy for 1 minute and on mininal energy for 10 minutes. 4.Stop heating once the buffer boils.5.Take the contianer  out of the microwave, and let it cool down for 15-30 minutes to room temprature. Do not transfer the slide directly from the boild buffer to other fluids at room temprature. Wait until proper cool down.
?. Bench, room temprature:Wash in a new 0.05% Tween-20 in PBS for 5 minutes.Wash again in a new 0.05% Tween-20 in PBS for 5 minutes.Take the slide out of the Tween PBS container, and put in the slide staining  box.Prepare blocking solution - 3% normal serum of goat/sheep/donkey/other in PBS (make sure it's a different species from the secondary antibody).Gently drop 250-300 ul the blocking solution on the slide.Cover the slide with a paraffin film and incubate  for 60 minutes.Remove the paraffin film from the slide. Gently tap the slide on an paper towel in order to dry it.
?. Bench, 4 °C:Add 150-200 ul of a primary antibody diluted in a blocking solutionCover with an new paraffin film. Incubate  for 60 minutes at room temprature or  4 °C. At 4°C, the antibody can stay on the slide over-night.
?. Bench, room temprature:Remove the paraffin film.Wash in  0.05% Tween-20 in PBS for 5 minutes.Wash again in a new 0.05% Tween-20 in PBS for 5 minutes.Wash for a thrid time in new 0.05% Tween-20 in PBS for 5 minutes.Add 150-200 ul of Secondary Ab diluted in  a  blocking solutionCover with paraffin film and incubate  60 minutes in room temprature.Wash in  0.05% Tween-20 in PBS for 5 minutes.Wash again in  a new 0.05% Tween-20 in PBS for 5 minutes.Wash for a thrid time in a new 0.05% Tween-20 in PBS for 5 minutes.Wash with DDW for 2 minutes
?. Stain with DAPI (1:2000 in DDW) for 2 minutes.1.Wash with DDW for 5 minutes.2.Wash in a new 0.05% Tween-20 in PBS for 5 minutes.3. Gently tap the slide on an paper towel in order to dry it
?. Proceed to microscopy analysis
?. Cover slides1.Add a few (2-3) drops of a water-based glue2. Cover slide with cover-slip3. Cover slide with an aluminium foil and let them dry over night
TITLE: Protocol of preparation of horseradish peroxidase (HRP) conjugated to anti-human IgG to be used as secondary antibody in immunoassays. 
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Peroxidase labeled anti-human IgG is a conjugated secondary antibody that can be used to detect specific antibodies against millions of antigens in humans. This reagent is usually used in enzyme-linked immunosorbent assay, immunoblot analysis and dot blot [1,2]. </div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.</span><span style = "font-style:italic;">J Pharm Biomed Anal</span><span>. 2013;72:208-215. doi:10.1016/j.jpba.2012.08.023</span></div><div class = "text-block">2.Justiz Vaillant AA, McFarlane-Anderson N, Akpaka PE, Smikle MP, Ramirez N, et al. (2013) Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals. J Chromat Separation Techniq 4: 181. doi:10.4172/2157- 7064.1000181  </div></div>

[STEPS]
?. Human IgG can be isolated from human serum or plasma by Protein-A agarose affinity chromatography. To develop the anti-human IgG a laboratory or farm animal is usually immunized and then, the anti-IgG is isolated. The Mancini test can estimate the anti-human IgG concentration.
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500µg/ml of anti-human IgG with 500 µg of the  mix of horseradish peroxidase-sodium periodate. The mixture is incubated for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
?. The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
?. Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The peroxidase conjugated to anti-human IgG is then stored at -20°C until further used.
TITLE: General proteomics FASP (Filter-Aided Sample Preparation)
AUTHORS: Jacob   Waldbauer, Lichun Zhang

[DESCRIPTION]
Spin-filter based protein digestion and purification for bottom-up proteomics

[BEFORE_START]
Passivate Vivacon filter unit (Satorius Vivicon 500, 30,000 MWCO) and collection tube overnight in 5% (v/v) Tween-20 in MilliQ water. The next day, rinse with nanopure water until no suds, then rinse 3x with LC-MS water.

[GUIDELINES]
It is important that nearly all liquid passes through spin filters after each centrifugation step. If a substantial amount of liquid (more than 75 μL) remains on filter after 10 minutes of centrifugation, spin for longer. 

[STEPS]
SECTION: Day 1
1. Prepare fresh buffers in LC-MS water: 
Exchange buffer: 8M urea, 0.2% (w/v) deoxycholate, 1M ammonium bicarbonate (pH 8)
Digestion buffer: 0.2% (w/v) deoxycholate, 50 mM ammonium bicarbonate (pH 8)
Trypsin dissolution/peptide recovery buffer: 50 mM ammonium bicarbonate (pH 8)
SECTION: Day 1
2. Dispense 25-50μL (depending on concentration) reduced, alkylated protein extract to filter unit.
SECTION: Day 1
4. Spin at 14,000g for 10 min, discard filtrate.
SECTION: Day 1
5. Repeat steps 3 and 4 three more times.
SECTION: Day 1
6. Wash filter unit with 200 μL digestion buffer, and spin at 14,000g for 10 min. Do this a total of 2 times.
SECTION: Day 1
3. Fill filter units with 200 μL exchange buffer, mix with pipet.
SECTION: Day 1
7. Transfer filter unit to passivated collection tube.
SECTION: Day 1
8. Add 110 μL digestion buffer and desired amount of trypsin (~1:50 trypsin/protein), dissolved in 50 mM ammonium bicarbonate, keeping trypsin on ice or at 4° C. Incubate at 37° C overnight. Parafilming tubes is recommended to prevent evaporation.
SECTION: Day 2
9. Remove parafilm if applicable and centrifuge tubes at 14,000g for 10 min. Do not discard filtrate!
SECTION: Day 2
10. Add 50 μL of peptide recovery buffer to filters, spin at 14,000g for 10 min. Do this step a total of 2 times.
SECTION: Day 2
11. Transfer the filtrate to a LoBind tube.
SECTION: Day 2
12. Add 900μL ethyl acetate and 2.5 μL TFA to filtrate, vortex. Precipitate may be visible.
SECTION: Day 2
13. Sonicate in a bath sonicator for 10s, and centrifuge at 16,000g for 10 min.
SECTION: Day 2
14. Carefully remove upper organic layer with a pipet without disturbing phase boundary.
SECTION: Day 2
15. Repeat steps 12, 13 and 14 two more times (Note: Only add 900μL ethyl acetate, no more TFA)
SECTION: Day 2
16. Place sample uncovered in Thermomixer and dry at 60° C for 5-10 minutes until ethyl acetate is gone.
SECTION: Day 2
17. Freeze sample solid at -80° C, Centrivap to dryness.
TITLE: Planetary Dominos - How microbes drive biogeochemical cycles
AUTHORS: Nadia Szeinbaum, Abbigail Johnson, Thomas Swofford

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the step to assemble your own Planetary Domino. </div><div class = "text-block"><span>We designed this at Georgia Tech for an Astrobiology outreach event called "</span><a href="https://www.facebook.com/events/1731095390302446/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">An Evening of Wonder</span></a><span>", which we organized as part of </span><a href="http://abgradcon.org/old_sites/abgradcon2018/index.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">AbGradCon2018</span></a><span>. The goal of this activity was to introduce how microorganisms drive many geochemical cycles and how they are interconnected. Each domino piece corresponds to a bacerial functional role/"trophic" role (i.e., aerobic heterotrophs, anaerobic metal reducers, etc.) contains substrates on one side and products on the other. The idea is to find how they can be connected, like pieces of a domino.   </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div>

[STEPS]
?. Print this file into a poster, cut each piece and add an adhesive magnet. These will be the domino pieces, and they are now able to stick to any magnetic surface. The pieces here are enough for at least three different sets, as they repeat. The design was created by Image Alleviation, in collaboration with us, specifically for this project.
?. After a brief introduction about microbes in the environment (good vs. bad, or biogeochemical cycles, depending on the audience), ask your audience to get into groups or one by one add pieces of the domino to the board. It is key is to let them discuss and ask questions, and only if necessary explain why certain pieces might not go together, etc.
TITLE: Cell lysis and extraction of total protein from Synechocystis sp. PCC 6803
AUTHORS: Anna Behle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cell lysis and extraction of total protein. Soluble and insoluble fractions can be further separated. Total protein can be then used for quantification and Western Blot analysis.</div></div>

[STEPS]
?. [Before starting]
Prepare 1x TB (thylakoid-buffer).  ABC1ComponentMolecular weight (g/mol)Concentration2HEPES / NaOH, pH = 7238,350 mM3MgCl25 mM4CaCl225 mM5(optional: glycerol)10 % (v/v)To 10 mL 1x TB: Add 1 tablet protease-inhibitor (complete ULTRA Tablets, Mini, EDTA-free, EASYpack by Roche)
ABC1ComponentMolecular weight (g/mol)Concentration2HEPES / NaOH, pH = 7238,350 mM3MgCl25 mM4CaCl225 mM5(optional: glycerol)10 % (v/v)
?. [Culturing]
Grow cyanobacteria to an OD750 of 0.5 - 1.0.
?. [Sampling]
Sample 10-20 mL of cyanobacterial culture.Pellet the cells at 4500 rpm, 4 ˚C.
?. [Cell lysis]
Work on ice.Resuspend cell pellet in 200 µL 1x TP. Add an equal volume of glass beads  (0.1 mm, 0.25 mm 1:1)Disrupt cells in Precellys homogenizer.Program: 4000 rpm, 2x 30 sec, 15sec pause, then back on ice
Make sure to use tubes suited for disruption in homogenizer - some tubes will break and you may lose your sample!
?. [Cell lysis]
Centrifuge (4 ˚C, 4000 rpm, 5 min), transfer supernatant to a fresh tube.Wash glass beads by adding 200 µL 1x TP. Repeat cell lysis step in Precellys.
?. [Cell lysis]
Centrifuge (4 ˚C, 4000 rpm, 5 min), add supernatant to the supernatant from step 5.
?. [Separation of membrane fraction from soluble protein]
Centrifuge extract at maximum speed for 30 min to 1 h, 4 ˚C. Soluble protein supernatant will appear blue, membrane pellet green.Check absorption spectrum of supernatant (chlorophyll peak at 665 nm should be completely gone).