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Proteomics Facility

Helpful Information

1. Gel Shipping Instructions Guidelines
2. Sequence-Quality SDS-Gel Guidelines
3. Web Links
4. Publications

1. GEL SHIPPING INSTRUCTIONS

Please note that we require that the entire gel be sent to us. We strongly recommend the gel be packaged and shipped per the instructions noted below. Gels that are improperly packaged may break in transit and cannot be used for analysis.

Gel(s) that are not securely packaged prior to shipment usually are severely damaged or destroyed during shipment due to abuse of the package by the shipper. We have found the following packaging technique to be successful (gel arrives intact):

  1. Take a picture or make a photocopy of the gel, mark on the copy the bands of interest, and include this with the submission sheet in a separate plastic bag in the shipping box—packed outside the ice (see further instructions regarding the use of ice).

  2. Place the gel in a watertight Ziploc® plastic bag after draining all excess water from the gel. The gel should remain moist with only a few drops of water in the bag. The gel should not be floating in the bag.

  3. Cut 2 pieces of cardboard slightly larger than the size of the bag containing the gel. Place the gel and bag between the cardboard covers and tape all sides.

  4. Securely wrap the cardboard/gel+bag/cardboard sandwich with bubble wrap.

  5. Place the entire bubble-wrapped sample on top of or near the top of loose, wet ice in a Styrofoam™ shipping container and send to us by the overnight courier of your choice. DO NOT use icepacks or pack bubble-wrapped sample between layers of wet ice, as this may freeze the gel and result in breakage.

  6. Send an email message to Kaye D. Speicher (not the website) the day the package is shipped. We will alert you if we do not receive the package by 4 p.m. the following day. Do not ship packages on a Friday afternoon for Saturday or other weekend/holiday delivery. 

SHIP TO: Wistar Proteomics Facility 
Attn: Kaye D. Speicher 
3601 Spruce St., Room 154 
Philadelphia, Pa. 19104-4268

2. SEQUENCE-QUALITY SDS-GEL GUIDELINES

Precautions and Changes to Laemmli Gels for MS or Sequence Analysis

Preparing Samples and Gels

  1. Maximize protein concentration in the gel. Mini Gels (1.0 mm thick) are preferred unless the higher resolution of a full-size gel is needed.

  2. Select a gel concentration that will give a sharp, tight band for the protein of interest, preferably with an Rf between 0.3 and 0.7. Use high-quality, pre-made gels such as Invitrogen™ NuPAGE® gels, and follow manufacturer guidelines or those shown below.

  3. Use electrophoresis reagents and solvents of highest purity (we use Bio-Rad®).

  4. Filter gel solutions (0.2 mM), except running buffer, and store at 4ºC, except SDS (R.T). Store solutions no more than one month.

  5. Solubilize samples using 2X or 5X solubilizing buffer containing sucrose or glycerol. DO NOT USE UREA!

  6. Do not heat samples excessively. If higher temperatures are needed to properly solubilize sample, minimize as much as possible, e.g. 1-2 minutes at 80-90ºC.

  7. If the gel has been made in-house, let the completely cast gel including the polymerized stacker stand submerged in Milli-Q® water for at least 24 hours but, no more than 48 hours at room temperature prior to use.

  8. Add 11.4 mg/L (0.1 mM) thioglycolate to the upper chamber buffer prior to electrophoresis, unless non-reducing conditions are needed.

  9. Follow manufacturer's guidelines when using precast gels.

Separation and Staining of Gels for In-Gel Digestions

  1. Avoid excessive heating (>25ºC) during electrophoresis.

  2. DO NOT run bromophenol blue dye front off the bottom of the gel.  This will not improve the separation.

  3. After electrophoresis immediately stain with either a),  b),
    or c): 

    a) Coomassie® blue R-250 30-60 minutes. Destain 1-4 hours maximum (background does not need to be completely clear). Rinse in high-quality water (such as Milli-Q) for 1 hour.

    b) Invitrogen colloidal Coomassie blue G-250 (order # LC6025 - follow manufacturer's guidelines for staining and destaining).

    c) Invitrogen Silverquest™ (order # LC6070 - follow manufacturer's guidelines for staining and destaining).

  4. Seal in Ziploc® bag and store at 4ºC (add only a few drops of water, gel should not be floating).

  5. The entire gel must be sent to us; follow "Gel Shipping Instructions."

  6. Include photocopy or picture of gel with desired band(s) for digestion clearly marked on the photocopy or picture.

General Gel Comments

- Urea decomposes readily to form cyanate, which will modify your protein at higher temperatures and pHs above 7. It is advisable to eliminate urea from your protocol when doing gels for sequence. The use of urea for isoelectrofocusing gels as a first step in 2D gels requires extra care to minimize protein modifications and proteins with pHs > 7 will probably be N-terminally blocked during isofocusing.

- Heating during sample prep or electrophoresis may contribute to chemical modifications of your protein.

- Conditioning “home made” gels, including the stacking gel, for 24-48 hours at room temperature prior to electrophoresis helps to eliminate free radicals.

- Thioglycolate scavenges free radicals and oxidants left in gel.

- DO NOT run the tracking dye off the gel! Instead, change the gel %, if needed, for optimal separation results.

3.USEFUL WEB LINKS

BLAST
Entrez
ExPaSy
ProFound
Adobe®
MASCOT®
MS-FIT

4. PUBLICATIONS

1. Mozdzanowski, J. and Speicher, D. W. 1990. Quantitative electrotransfer of proteins from polyacrylamide gels onto PVDF membranes. In: Current Research in Protein Chemistry (J. Villafranca, ed.), Academic Press, pp. 87-94.

2. Harper, S.L., and Speicher, D.W. 1997. Expression, isolation and protease cleavage of GST fusion proteins in E. coli. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 6.6.1-9.

3. Harper, S., Mozdzanowski, J., and Speicher, D.W. 1998. Two-dimensional gel electrophoresis. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York, NY. pp. 10.4.1-36.

4. Speicher, D.W. 1998. Characterization of protein primary structure. In: Characterization of Biotechnology Pharmaceutical Products. Development of Biological Standards. Karger. Basel Vol. 96:25-26.

5. Mische, S., Hellman, U., Speicher, D., and Williams, K. 1999. Internal protein sequencing. In: Encyclopedia of Bioprocess Technology. John Wiley & Sons, Inc. New York. pp. 2099-2102.

6. Begg, G.E. and Speicher, D.W. 1999. Mass spectrometry detection and reduction of disulfide adducts between reducing agents and recombinant proteins with highly reactive cysteines. J. Biomolecular Techniques 10:17-20. 

7. Zuo, X. and Speicher, D.W. 2000. Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:3035-3047.

8. Zuo, X. and Speicher, D.W. 2000. A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal. Biochem. 284 (2):266-278.

9. Speicher, K.D., Kolbas, O., Harper, S., and Speicher, D.W.  2000.  Systematic analysis of peptide recoveries from in-gel digestions for femtomole protein identifications in proteome studies.  J. Biomol. Tech., 11:74-86.

10. Chong, J.M. and Speicher, D.W.  2001.  Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, Ep-CAM).  J. Biol. Chem., 276:5804-5813.

11. Carl, P., Kwok, C.H., Manderson, G., Speicher, D.W., and Discher, D.E. 2001.  Forced unfolding modulated by disulfide bonds in the immunoglobulin domains of the cell adhesion molecule Mel-CAM.  Proc. Natl. Acad. Sci. USA, 98:1565-1570.

12. Begg, G.E, Harper, S.L., and Speicher, D.W.  2001. Characterizing recombinant proteins using HPLC gel filtration and mass spectrometry.  In:  Current Protocols in Protein Science.  John Wiley & Sons, Inc., New York, NY. pp. 7.10.1-15.

13. Harper, S., Mozdzanowski, J., Speicher, D.W. 2001. Two-dimensional gel electrophoresis. Curr Protoc Protein Sci. pp 10: 4.

14. Ali-Khan, N., Zuo, X., and Speicher, D.W. 2002.  Overview of proteome analysis. In: Current Protocols in Protein Science.  John Wiley & Sons, Inc., New York, NY. pp. 22.1.1-19.

15. Zuo, X. and Speicher, D.W. 2002.  Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional gels.  Proteomics, 2:58-68.

16. Chong, J.M., Uren, A., Rubin, J.S., and Speicher, D.W. 2002.  Disulfide bond assignments of secreted Frizzled- related protein-1 provide insights about frizzled homology and netrin modules. J. Biol. Chem., 277:5134-5144.

17. Zuo, X., Hembach, P., Echan, L., and Speicher D.W. 2002.  Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.  Journal of Chromatography B, 782:253-265.

18. Speicher, D.W. 2004.  Overview of proteome analysis. In: Proteome Analysis: Interpreting the Genome (D. Speicher, ed.).  Elsevier, The Netherlands, pp. 1-18.

19. Speicher, D.W., Lee, K., Tang, H.-Y., Echan, L., Ali-Khan, N., Zuo, X., and Hembach, P. 2004. Current challenges in proteomics: Mining low abundance proteins and expanding protein profiling capacities. In: Advances in Mass Spectrometry, Vol. 16 (Ashcroft, A.E., Brenton, G, and Monaghan, J.J., ed.) pp. 37-57.

20. Tang, H-Y. and Speicher, D.W. 2004.  Determination of disulfide bond linkages in proteins.  In: Current Protocols in Protein Science.  John Wiley & Sons, Inc., New York, NY.

21. Murphy, S.C., Samuel, B.J., Harrison, T., Speicher, K.D., Speicher, D.W., Reid, M.E, Prohaska, R., Low, P.S., Tanner, M., Mohandas, N., Haldar, K. 2004.  Erythrocyte detergent resistant membrane proteins: their characterization and selective uptake during malarial infection.  Blood, 103:1920-1928.

22. Tang, H.-Y., Speicher, D.W. 2004. In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner.  Biochemistry, 43:4251-4262.

23. Tang, H-Y. and Speicher, D.W. 2004.  Identification of alternative products and optimization of 2-nitro-5-thiocyanobenzoic acid cyanylation and cleavage at cysteine residues.  Anal. Biochem., 334:48-61.

24. Echan, L., Tang, H-Y., Ali-Khan, N., Lee, K. and Speicher, D.W. 2005.  Depletion of multiple high - abundance proteins improves detection of lower abundance proteins in human serum and plasma.  Proteomics, 5:3292-3303.

25. Rai, A.J., Gelfand, C.A., Haywood, B.C., Warunek, D., Skobe, C., Schuchard, M.D., Mehigh, R.J., Cockrill, S.L., Scott, G.B.I., Tammen, H., Schulz-Knappe, P., Speicher, D.W., Siest, G., Vitzthum, F., Haab, B.B. and Chan, D.W. 2005.  Human Proteome Organization-Plasma Proteome Project Specimen Collection and Handling: Towards the standardization of parameters for plasma proteome samples.  Proteomics, 5:3262-3277.

26. Tang, H-Y., Ali-Khan, N., Echan, L.A., Levenkova, N., Rux, J.J. and Speicher, D.W. 2005.  A novel 4-dimensional strategy combining protein and peptide separation methods enables detection of low abundance proteins for comprehensive profiling in human plasma and serum proteomics.  Proteomics, 5:3329-3342.

27. Tang, H-Y. and Speicher, D.W.  2005. Complex proteome prefractionation using microscale solution isoelectrofocusing to improve protein coverage and detection of low abundance proteins.  Expert Review of Proteomics, 2:295-306.

28. States, D.J., Omenn, G.S., Blackwell, T.W., Damian, F., Eng, J., Speicher, D.W. and Hanash, S. M. 2006.  Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study.  Nature Biotechnology, 24:333-338.

29. Fischer, R.S., Yarmola, E.G., Weber, K.L., Speicher, K.D., Speicher, D.W., Bubb, M.R. and Fowler, V.M. 2006.  Tropomodulin 3 binds to actin monomers.  J Biol Chem., 281:36454-36465.

30. Hoffman, S.A., Joo, W-A., Echan, L.A. and Speicher, D.W. 2007.  Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma.   Journal of Chromatography B, 849:43-52.

31. Liu, Q., Tan, G., Levenkova, N., Li, T., Pugh, Jr., E.N., Rux, J., Speicher, D.W. and Pierce, E.A. 2007.  The proteome of the mouse photoreceptor sensory cilium complex.  Mol Cell Proteomics, 6:1299-1317.

32. Johnson, C.P., Tang, H-Y., Carag, C., Speicher, D.W. and Discher, D.E. 2007.  Forced unfolding of proteins within cells.  Science, 317:663-666.

33. Li, D., Tang, H.-Y, and Speicher, D.W. 2008. A structural model of the erythrocyte spectrin heterodimer initiation site determined using homology modeling and chemical crosslinking.  J. Biol. Chem. 283:1553-1562.

34. Han, M-J., Herlyn, M., Fisher, A.B. and Speicher, D.W. 2008.  Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts.  Electrophoresis.  29:695-705.

35. Han, M.J., Speicher, D.W. 2008. Microscale isoelectric focusing in solution: a method for comprehensive and quantitative proteome analysis using 1-D and 2-D DIGE combined with MicroSol IEF prefractionation. Methods Mol Biol. 424: 241-256.

36. Keene, S.D., Greco, T.M., Parastatidis, I., Lee, S-H., Hughes, E.G., Balice-Gordon, R.J., Speicher, D.W. and Ischiropoulos, H. 2009.  Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome. Proteomics, Jan. 8, Proteomics. 9:768-82.

37. Wu, Y., Feinstein, S.I., Manevich, Y., Chowdhury, I., Pak, J.H., Kazi, A., Dodia, C., Speicher, D.W., and Fisher, A.B. 2009.   Mitogen activated protein kinase-mediated phosphorylation of peroxiredoxin 6 regulates its phospholipase A2 activity.  Biochem J. Jan. 14, 2009. Epub ahead of print.

 

  

 

 

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