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extracting-cholesterol-and-lipids-from-cells.rst

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+Extracting cholesterol and lipids from cells
+========================================================================================================
+
+.. sectionauthor:: mfitzp <martin.fitzpatrick@gmail.com>
+
+Contributed by `Martin Fitzpatrick <http://martinfitzpatrick.name/>`__, University of Birmingham, United Kingdom
+
+This a general method that may not work for all cell/tissue samples - tweak the solvent volumes per quantity of cells, centrifugation time and sonication may be applied for samples that are difficult to disrupt.  For chloroform - handle under a chemical safety hood.
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+- Homogenize 1 x 10e6 cells or ~10 mg tissue into either 200 uL chloroform-methanol (v/v 2:1) or 200 uL hexane-isopropanol (v/v 3:2).
+
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+- Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge.
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+- Transfer the organic phase to a clean tube and vacuum dry.  Store the material in the freezer (<20oC), desiccated and protected from air, i.e., under anaerobic conditions to minimize oxidation.
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+- Re-dissolve the vacuum-dried lipids/cholesterol into a suitable assay buffer prior to use.
+
+
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+
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+References
+----------
+
+
+FOLCH J, LEES M, SLOANE STANLEY GH `A simple method for the isolation and purification of total lipides from animal tissues. <http://www.ncbi.nlm.nih.gov/pubmed/13428781>`_ *J Biol Chem* (1957)
+`pmid:13428781 <http://www.ncbi.nlm.nih.gov/pubmed/13428781>`_
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+

in-gel-tryptic-digest-for-protein-id-by-ms.rst

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+In-gel Tryptic Digest for Protein ID by MS
+========================================================================================================
+
+.. sectionauthor:: timothymitchison <timothy_mitchison@hms.harvard.edu>
+
+Contributed by `Timothy Mitchison <https://sysbio.med.harvard.edu/facultys/timothy-j-mitchison-phd/>`__, Harvard Medical School, Boston, MA, United States
+
+In-gel Tryptic Digest for Protein ID by Mass Spectrometry
+(David Miyamoto, 2/12/2002). This protocol is based on Shevchenko A, Wilm M, Vorm O, & Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 1996, 68:850-8. I have used it with success on both Coomassie and silver-stained gel bands.
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+The procedure includes reduction and acetamidation steps that may be skipped if desired. For heavily stained Coomassie bands, it is helpful to wash gel pieces for 1 hr in 100 mM NH4HCO3 prior to dehydrating with acetonitrile (step 2).
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+Method
+------
+
+- Excise band from Coomassie or silver stained gel. Cut gel band into 1 mm cubes using clean razor blade on a clean glass surface. Transfer to an Eppendorf tube.
+
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+- Remove excess water with pipet. Add 25-35 µL acetonitrile to tube to cover gel pieces. Incubate 10 minutes at RT to dehydrate and shrink gel pieces.
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+- Remove acetonitrile with pipet. Speed-vac to dryness for 10 minutes.
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+- Swell gel particles in 150 µL 10 mM DTT in 100 mM NH4HCO3. Incubate 1 hour at 56°C.
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+- Cool to RT. Replace DTT solution with 150 µL 55 mM iodoacetamide in 100 mM NH4HCO3. Incubate 45 minutes at RT in the dark with occasional vortexing.
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+- Remove solution and wash gel pieces with 150 µL 100 mM NH4HCO3. Incubate 10 minutes at RT.
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+- Remove NH4HCO3 solution with pipet. Add 150 µL acetonitrile to dehydrate gel pieces. Incubate 10 minutes at RT.
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+- Repeat wash steps 6 through 7. Remove acetonitrile and speed-vac to dryness for 10 minutes.
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+- Place tubes in ice water bath and swell gel particles in 25-35 µL digestion buffer. Incubate 45 minutes in ice water bath. Digestion buffer consists of 12.5 ng/µL trypsin (Promega sequence-grade modified porcine trypsin, Cat. #V511A) in 50 mM NH4HCO3. To make the digestion buffer, dissolve 20 µg Promega trypsin in 80 µL Promega trypsin buffer solution (50 mM acetic acid), and dilute with 50 mM NH4HCO3 to 12.5 ng/µL.
+
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+- Remove trypsin-containing buffer. Add 5-10 µL 50 mM NH4HCO3 without trypsin to keep pieces wet during cleavage. Incubate o/n at 37°C.
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+- Spin 1' at 14,000 rpm to spin down gel pieces. Save supernatent in a separate PCR tube.
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+- Add 20 µL 20 mM NH4HCO3 to cover gel pieces. Incubate 10 minutes at RT. Transfer supernatent to the PCR tube from step 11.
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+- Add 25 µL 5% formic acid, 50% acetonitrile to the gel pieces. Incubate 20 minutes at RT.
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+- Spin 1' at 14,000 rpm. Remove formic acid/acenonitrile solution and save in the same PCR tube from step 11.
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+- Repeat formic acid extraction (steps 13 through 14) twice more.
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+- Dry PCR tube in speed-vac to complete dryness. Store at -20°C until analysis.
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+References
+----------
+
+
+Shevchenko A, Wilm M, Vorm O, Mann M `Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. <http://www.ncbi.nlm.nih.gov/pubmed/8779443>`_ *Anal Chem* (1996)
+`pmid:8779443 <http://www.ncbi.nlm.nih.gov/pubmed/8779443>`_
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+

purification-of-6x-his-proteins.rst

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+Purification of 6X HIS Proteins
+========================================================================================================
+
+.. sectionauthor:: ianchinsang <chinsang@queensu.ca>
+
+Contributed by `Ian Chin-Sang <http://post.queensu.ca/~chinsang/>`__, Queens University, ON, Canada
+
+Purification of 6XHIS proteins with cell extraction buffer
+
+
+.. figure:: /images/method/1444/384px-Ni_column.JPG
+   :alt: method/1444/384px-Ni_column.JPG
+
+
+
+
+
+
+Requirements
+------------
+Extraction buffer: 10mM imidazole, 500mM NaCl, 50mM NaH2PO4, pH8.0, (Optional components:  0.5mM TCEP, 1x protease inhibitor cocktail -Complete PI EDTA free tablets; Benzonase Nuclease HC,3 µl per 60ml extraction buffer).
+
+
+Method
+------
+
+- Spin cells harboring 6XHIS proteins in large bottles in Beckman Centrifuge 6000rpm 15 minutes.
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+- Dump supernatant
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+- Resuspend in 3ml Cell extraction buffer.
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+- Grind protein in mortar and pestle plus liquid Nitrogen. (at least 10 minutes.) until becomes a fine powder.
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+- Transfer frozen powder to 15ml conical tube and bring volume up to 10 ml.
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+- Transfer approx 1ml to microcentrifuge tube and spin for 15 minutes at full speed 4C.
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+- While spinning pre-equilibrate Ni-NTA resin with extraction buffer by washing 3X in extraction buffer and resuspend in 50% slurry.
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+- Pool all the supernatants from step 6 into a 15ml conical (save 100ul of supernatant =Load) and add about 300 ul of  Ni-NTA resin to the supernatant. Save some of the pellet (= insoluble fraction).
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+- Bind 6XHIS protein to resin for 20min to 1 hour room temp.  Spin and save 100ul of the supernatant= unbound fraction.
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+- Wash resin 5 times with extraction buffer (5ml each time).
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+- Elute with low pH elution buffer (1ml each elution).  Elute 5 X.
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+This method is based, with permission, on an original protocol available `here <http://130.15.90.245/purification_of_6x_his_proteins.htm>`_.