Proteomics Services Core

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Guidelines for Submitting Samples

Always consult with the Proteomics Core manager prior to submitting (submission form) your samples for analysis. This will ensure that we understand your specific needs before submission of samples.

  • Sample types: We accept plasma or tissue samples for analysis. Please consult with us for submission of other sample types.
  • Investigators should ensure that samples are free of precipitates or large particles as this may clog or dirty the machine.
  • Be aware that certain buffers for homogenization are not compatible with the mass spec. Detergents that can be detrimental to system include the following:
    • Incompatible detergents
      • Nonidet P-40 (which can no longer be purchased; Sigma is substituting CA- Igepal 630)DSC_0409.JPG
      • Triton X-100 (or any derivative)
      • Igepal/PEG (any derivative)
      • Brij-35 (or any derivative)
      • Tween-20 OTG
      • CHAPSO
      • Type NP40/NP40 alternative
    • Incompatible buffers
      • HEPES, PBS, MES, MOPS, and Tris
        Some detergents can be separated from the sample by standard SDS-PAGE or TCA - cold acetone precipitation. However, Triton-X and Tween-20 cannot be used under any circumstances. These cannot be removed from your sample using dilution, washing, detergent spin columns, or SDS-PAGE.
    • Mass spec-friendly detergents
      • 0.05%-1% SDS
      • 0.05%-0.5% CHAPS (although not recommended)
      • N-octyl-β-glucopyranoside
      • PPS Silent Surfactant (acid-cleavable detergent)
      • Protea biosciences (anionic, zwitterionic,
        or cationic acid labile detergents)
      • Big CHAP deoxy (merck)
      • ASB series (EMD chemicals)
      • Sodium deoxycholate
    • Mass spec-friendly buffers
      • Ammonium acetate
      • Ammonium bicarbonate
      • Ammonium formate
    • Sample lysis buffers
      • Reagent 4 (Sigma C0356)
      • 8M urea with 1M ammonium bicarbonate
  • We will analyze samples in bulk to cut costs. Thus, we can advise you when your samples will be assay.
  • By checking with us first, we can also ensure that the needed calibrators, controls and reagents are on hand.

Sample preparation

  • Complete the rational for service form and sample submission form specific for proteomic analysis.
  • A minimum of 600ug of tissue (recommended buffer volume 90-200ul) or 15ul of plasma is required for analysis. A Bradford assay will be performed on submitted tissue samples and any samples with low protein will not be processed.
  • Example extraction methods:

    Reagent 4:

    1x Protease Inhibitor Cocktail (Roche cOmplete Mini with EDTA, ordered from Fisher #50-720-4060):  Solubilize 1 tablet in 1 mL PBS to make a 10x solution. All buffers have to contain 1x PI final concentration.

    Reagent 4: urea, thiourea, Trizma, detergent C7BzO

    To dissolve: 1. Add 15 ml dH2O to bottle and swirl,
                      2. Heat to 30°C for 30-60 min, until dissolved, 
                      3. Use fresh or aliquot (1ml/tube) and store at -20°C


    1. Weigh sample, record weight on your lab-book, and place tissue in 5 ml round bottom tube (Fisher, Catalogue No. 14-956-3A).
    2. Add lyses buffer (Reagent 4 + 1x PI) to tube containing tissue and 16 µL per mg tissue (our homogenizer required a minimum of 250ul).
    3. Homogenize using PowerGen 1000 from Fisher Scientific or other homogenizer. Start the machine at level 1 and slowly increase until your sample is completely homogenized. (Do not increase above 4-5 in power or there will be too many bubbles.) If your tissue sample is small (10-20 mg) you may need to use the sonicator. Use 40% power and a 2-3sec/pulse and DO NOT allow your sample to heat, or you will denature your protein.
    4. Transfer to 1.5 ml tube and store at room temperature until all samples are homogenized. Centrifuge at 3000 rpm for 2 minutes at 4˚C to reduce the foam.
    5. Store at -80°C or use aliquot immediately for protein quantification by the Bradford Assay.

     8M Urea with 1M ammonium bicarbonate:

    1. Cut tissue into small pieces (1-3mm)
    2. Sonicate sectioned tissues until it disappears using 16 µL of lysis buffer per mg tissue (I recommend a minimum of 250ul). Sonicate tissues in 5 cycles, each with 30 second sonication and 1 minute cooling on ice at power setting of 40%. More sonication cycles may be needed to break down the tissue sections.
    3. Measure the protein concentration using BCA assay (Standard 0,25,125,250,500, 1000 µg/mL BSA, duplicates)
    4. Send 600ug to the core and save the rest of the proteins for future use.
    • For power analysis, it is recommended that 6 samples per group be ran and all samples of a study must be ran together.
    • Submit only the amount requested for your assays in a microcentrifuge tube (1.5ml of 0.5ml Eppendorf tube). The core does not have the capability to spin down samples provided in non-conventional tubes (screw-cap, plates, etc.). Stock samples with large volumes or samples provided in non-conventional tubes will be returned to the core user for proper aliquoting before the assay can be performed.
    • Each sheet should be species specific. Do not mix rat and mouse samples on the same sheet.