diff --git a/extracting-cholesterol-and-lipids-from-cells.rst b/extracting-cholesterol-and-lipids-from-cells.rst new file mode 100644 index 0000000..869e656 --- /dev/null +++ b/extracting-cholesterol-and-lipids-from-cells.rst @@ -0,0 +1,44 @@ +Extracting cholesterol and lipids from cells +======================================================================================================== + +.. sectionauthor:: mfitzp + +Contributed by `Martin Fitzpatrick `__, 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. + + + + + + + + +- 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). + + +- Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge. + + +- 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. + + +- Re-dissolve the vacuum-dried lipids/cholesterol into a suitable assay buffer prior to use. + + + + + +References +---------- + + +FOLCH J, LEES M, SLOANE STANLEY GH `A simple method for the isolation and purification of total lipides from animal tissues. `_ *J Biol Chem* (1957) +`pmid:13428781 `_ + + + + + + + diff --git a/in-gel-tryptic-digest-for-protein-id-by-ms.rst b/in-gel-tryptic-digest-for-protein-id-by-ms.rst new file mode 100644 index 0000000..a8dda9b --- /dev/null +++ b/in-gel-tryptic-digest-for-protein-id-by-ms.rst @@ -0,0 +1,87 @@ +In-gel Tryptic Digest for Protein ID by MS +======================================================================================================== + +.. sectionauthor:: timothymitchison + +Contributed by `Timothy Mitchison `__, 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. + + + + +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). + + + + + + +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. + + +- 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. + + +- Remove acetonitrile with pipet. Speed-vac to dryness for 10 minutes. + + +- Swell gel particles in 150 µL 10 mM DTT in 100 mM NH4HCO3. Incubate 1 hour at 56°C. + + +- 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. + + +- Remove solution and wash gel pieces with 150 µL 100 mM NH4HCO3. Incubate 10 minutes at RT. + + +- Remove NH4HCO3 solution with pipet. Add 150 µL acetonitrile to dehydrate gel pieces. Incubate 10 minutes at RT. + + +- Repeat wash steps 6 through 7. Remove acetonitrile and speed-vac to dryness for 10 minutes. + + +- 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. + + +- 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. + + +- Spin 1' at 14,000 rpm to spin down gel pieces. Save supernatent in a separate PCR tube. + + +- Add 20 µL 20 mM NH4HCO3 to cover gel pieces. Incubate 10 minutes at RT. Transfer supernatent to the PCR tube from step 11. + + +- Add 25 µL 5% formic acid, 50% acetonitrile to the gel pieces. Incubate 20 minutes at RT. + + +- Spin 1' at 14,000 rpm. Remove formic acid/acenonitrile solution and save in the same PCR tube from step 11. + + +- Repeat formic acid extraction (steps 13 through 14) twice more. + + +- Dry PCR tube in speed-vac to complete dryness. Store at -20°C until analysis. + + + + + +References +---------- + + +Shevchenko A, Wilm M, Vorm O, Mann M `Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. `_ *Anal Chem* (1996) +`pmid:8779443 `_ + + + + + + + diff --git a/purification-of-6x-his-proteins.rst b/purification-of-6x-his-proteins.rst new file mode 100644 index 0000000..23d6ea7 --- /dev/null +++ b/purification-of-6x-his-proteins.rst @@ -0,0 +1,65 @@ +Purification of 6X HIS Proteins +======================================================================================================== + +.. sectionauthor:: ianchinsang + +Contributed by `Ian Chin-Sang `__, 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. + + +- Dump supernatant + + +- Resuspend in 3ml Cell extraction buffer. + + +- Grind protein in mortar and pestle plus liquid Nitrogen. (at least 10 minutes.) until becomes a fine powder. + + +- Transfer frozen powder to 15ml conical tube and bring volume up to 10 ml. + + +- Transfer approx 1ml to microcentrifuge tube and spin for 15 minutes at full speed 4C. + + +- While spinning pre-equilibrate Ni-NTA resin with extraction buffer by washing 3X in extraction buffer and resuspend in 50% slurry. + + +- 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). + + +- Bind 6XHIS protein to resin for 20min to 1 hour room temp. Spin and save 100ul of the supernatant= unbound fraction. + + +- Wash resin 5 times with extraction buffer (5ml each time). + + +- Elute with low pH elution buffer (1ml each elution). Elute 5 X. + + + + + + + +This method is based, with permission, on an original protocol available `here `_.