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ProteoRed Multicentric Experiment 5 2D electrophoresis analysis

Salamanca, 16-17 March, 2010. ProteoRed Multicentric Experiment 5 2D electrophoresis analysis. Samples to be analyzed . Centers involved.

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ProteoRed Multicentric Experiment 5 2D electrophoresis analysis

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  1. Salamanca, 16-17 March, 2010 ProteoRed Multicentric Experiment 5 2D electrophoresis analysis

  2. Samplesto be analyzed. Centersinvolved A mixture of around 150 E. Coli proteins (identical in each sample). This mixture has been prepared by fractionation of the cytoplasmatic proteome of E.Coli. It contains soluble proteins, of a wide range of pI and Mw. Four spiked mammalian proteins: CYC_HORSE (Cytochrome C, Mw 12362), added at the ~ 1 pmol/ 1 mg total protein level. MYG_HORSE (Apomyoglobin, Mw 16952), at ~ 200 fmol / 1 mg total protein ALDOA_RABIT (Aldolase, Mw 39212), at ~ 25 fmol / 1 mg total protein ALBU_BOVIN (Serum albumin, Mw 66430), at ~ 1 fmol / 1 mg total protein  These four proteins have been spiked in different amounts in samples A and B, with ratios ranging from 1.5:1 to 5:1 between the two samples. Results available from HUVH, CIB, IIB-UAB and CIMA.

  3. Experimental procedure • Data (MIAPE) are available from HUVH and CIMA. • Sample preparation and application: • Sample buffer: 7M urea, 2M thiourea, 4% CHAPS, 120/40 mM DTT; ampholites pH range 3-10 (1%) or 3-11 (2%); 1,2% destreak used in HUVH • First dimension: • Sample loading: cup loading, 150 mg HUVH and 9 mg CIMA. • IPG strips pH range 3-10 or 3-11 NL. • Second dimension: • 12,5% acrylamide gels (30%/0.8% or 40%/2.6% acrylamide/bis-acrylamide) • Inter dimension treatment: No differences. • Image capture: • Typhoon 9400 or trio with similar settings. • Image analysis: • Same Spots v 3.3 or Decyder v 6.5. Automated mode.

  4. Gel images. Group 1 Gel N.72052 Gel N. 72051 Gel N. 72053

  5. Analysisoftheresults

  6. Quantificationofthespikedproteins

  7. Representative gel images Group 2 Group 1 Group 3 Group 4

  8. T-test 2.10E-05 Ratio -1.73 493 T-test 7.70E-06 Ratio -1.73 492

  9. 858 T-test 1.40e-06 Ratio -2.82 857 T-test 3.30e-06 Ratio -2.93

  10. 500 T-test 5.20E-05 Ratio -1.8 T-test 0.00062 Ratio -1.79 372

  11. 489 T-test 9.80E-06 Ratio -1.77 495 T-test 1.60E-05 Ratio -1.77

  12. 362 T-test 0.00014 Ratio -1.69 926 T-test 0.0089 Ratio -1.73

  13. Cuantificacion Cleanup Proteored muestras para Dige ug/ul BSA A 570

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