Protein Sample Preparation

Protein Sample Preparation

 

Cell lysis

  • Lyse cells in 0.5M TEAB 0.05% SDS  (Protease and Phosphatase inhibitor cocktail and Benzonase) Incubate for 30 mins at 4°C. Vortex briefly every 10 minutes. Check sample is ‘clear’ of DNA. (Lyse in 50µl per 106 cells)
  • Centrifuge cell lysate at 10,000 g at 4°C for 10 mins.
  • Measure protein concentration using the Bradford protein assay

Nuclear /cytoplasmic extraction

Protocol for 8-9 x 106 (1x 107) cells using the Active Motif nuclear extraction kit

    • Wash buffer: per cell line

      0.8 ml of 10 X PBS,  6.8 mls water and 0.4mls phosphatase inhibitors  OR Hanks if interested in phosphorylation

    • Hypotonic buffer: per cell line

       50µl of 10 x buffer, 450µl of water

    • Lysis buffer: per cell line

      50µl of 0.5M TEAB, 0.05% SDS and 0.5µl of protease inhibitor cocktail and 2µl/ml Benzonase

  • Harvest

    Wash cell twice in 4mls (6mls) of ice cold wash buffer and pellet.

  • Cytoplasmic fraction

    Gently resuspend in 500µl (750µ) of hypotonic buffer and transfer to prechilled eppendorf. Incubate on ice for 15 minutes. Add 25µl (37.5µl) of detergent and vortex for 10 seconds.

    Centrifuge at 14,000g for 30seconds.

    Transfer supernatant (cytoplasmic fraction) and use the pellet for nuclear fraction. Take care to remove all the supernatant.

  • Nuclear fraction

    Resuspend the nuclear pellet in 50µl (75µl) of TEAB lysis buffer. Vortex for 10seconds and incubate for 20 minutes on ice. Vortex for 30 seconds and centrifuge for 10minutes at 14000g. Transfer supernantant (nuclear fraction) into new eppendorf.

NOTE: Apparently the cytoplasmic lysis buffer contains Tris therefore unsuitable for direct iTRAQ labelling

Tryptic digest

  • Digest 100μg of protein for each cell line (volume as low as possible, 30µl?)
  • Reduce disulphide bonds by adding 0.1 volumes of `Protein reducing buffer' from the iTRAQ kit (50mM tris-(2-carboxyethyl)phosphine; TCEP). Vortex, pulse spin and incubate in heating block at 60°C for 1 hour.
  • Alkylate protein by addition of 0.05 volumes of `Cysteine blocking reagent' from the iTRAQ kit (200mM methylmethanethiosulphate; MMTS). Vortex, pulse spin and incubate at room temp for 10 minutes.
  • Digest protein by adding trypsin at a 10:1 substrate:enzyme ratio (i.e. 10ug trypsin per 100ug of protein). Lyophilised trypsin should be reconstituted in supplied buffer. Ensure that the final concentration of SDS is 0.05% or lower, add more 0.5M TEAB if necessary. Vortex to mix, pulse spin and incubate at 37°C in a water bath or incubator overnight.

iTRAQ labeling

  • iTRAQ reagents are unstable and hydrolyse very easily. Therefore repeated freeze thawing should be avoided. Please take contents out of the kit one at a time and as quickly as possible before returning the kits to -20°C to prevent iTRAQ reagent thawing out.
  • Dry down the tryptic digest in a SpeedVac to less than 40uL volume if necessary.
  • Take iTRAQ vials out of the freezer. Note the batch number, and the correction factors on the data sheet in the kit. Allow vials to thaw on the bench for 2-3 mins.
  • Spin iTRAQ vials to get all label to the bottom of the tube (reagent comes diluted in 5uL acetonitrile).
  • Add 40ul ethanol to the vial of iTRAQ reagent, and transfer to sample vial. Wash the iTRAQ reagent vial out with a further 30ul of ethanol and add this to the sample, resulting in a total of 70uL ethanol in the labeling reaction.
  • Repeat for each label/sample.
  • Vortex to mix, pulse spin and incubate on the bench for 1hour.
  • Pool the samples and dry down both samples in the SpeedVac. (Samples can be stored at -20°C at this stage if required).
  • Samples may form precipitates as addition of iTRAQ reagent may result in decreased solubility (large amount of precipitation indicative that proteins present ie failure of tryptic digest).

In-gel Trypsin Digestion

  • Excise spots from gel into methnol-washed eppendorfs. Spots can be stored in the fridge for at least two months. Ideally, gel should be chopped into small pieces.
  • For silver stained gel pieces, destain spots using a 1:1 mixture of 30mM K3Fe(CN)6  (10mg/mL) and 100mM Na2S2O3 (25mg/mL). Make fresh and filter before use. Use 50-70ml per gel piece, ensuring that gel is covered. If there is still brown colouring visible, replace destain with a freshly mixed solution for a further 5-10 mins.
  • For Coomassie Blue stained gel pieces, or for destained (now yellow) silver gel pieces, remove colour by (repeated) incubation in 200mM ammonium bicarbonate, 40% (v/v) acetonitrile.
  • Dry gel pieces with two to three washes in 50ml (ensuring gel is covered) 100% acetonitrile (15mins). Gel pieces should be completely opaque by the end of the third wash. During this step, gel pieces may clump together. Disaggregate with a pipette tip or using a methanol-wiped needle. Cool samples on ice for 10mins.
  • Thaw trypsin aliquot (stock at 500ng/uL) and keep on ice. Dilute from stock 1:10 in 100mM ammonium bicarbonate, 5% (v/v) acetonitrile and add 5ml to each sample. Pulse spin sample. If the gel piece is not covered, keep adding small amounts of trypsin and spinning until the gel piece is JUST covered. Leave samples on ice for 20-30mins to allow all of the enzyme solution to enter the gel.
  • Add 25ml ice-cold buffer (100mM ammonium bicarbonate, 5% (v/v) acetonitrile) to prevent drying out and incubate at 37°C overnight.
  • Peptide Extraction: Remove supernatant to a clean tube. Extract peptides from gel by adding 25ml 50% ACN with 0.1% formic acid. Vortex, and sonicate in water bath for 15 mins. Spin down gel pieces and pool supernatant with that from before. Dry pooled supernatants in SpeedVac. Store at -20ºC until analysis (500mM ammonium bicarbonate stock = 0.79g/20mL).