Helpful Information

Gel shipping instructions
Sequence-quality SDS-gel guidelines
Useful links
Publications

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 two 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 Hsin-Yao Tang the day the package is shipped. We will alert you if we do not receive the package by 4:00 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: Hsin-Yao Tang, Ph.D.
3601 Spruce St., Room 252 

Philadelphia, Pa. 19104-4268

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 µ), 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 “homemade” 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.

 

Useful links

BLAST - Search protein and nucleotide databases to identify sequence similarities.

EMBL - An alternative website that enables searching of a non-redundant database to identify sequence similarities.

Mascot® - A search engine that uses mass spectrometry data to identify proteins from sequence databases.

Entrez - Integrated access to biomedical literature, nucleotide and protein sequences, 3-D protein structures, complete genomes and population study data.

ExPaSy - A list of tools (websites) for proteomic type analysis. 

ProFound - A peptide mapping tool.

 

Publications

1. 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. Biomol. Tech. 10:17-20. PMID: 19499002. PMCID: PMC2291582.

2. 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. PMID: 19499040. PMCID: PMC2291619.

3. 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. PMID: 11080501.

4. 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. PMID: 11741940.

5. Yan, Y., Harper, S., Speicher, D.W., and Marmorstein, R. 2002. The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate. Nature Struct. Biol. 9:862-869. PMID: 12368900.

6. Hakimi, M.A., Dong, Y., Lane, W.S., Speicher, D.W., Shiekhattar, R. 2003. A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes. J. Biol. Chem. 278:7234-7239. PMID: 12493763.

7. Tang, H.-Y. and Speicher, D.W. 2004. In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner. Biochemistry. 43:4251-4262. PMID: 15065869.

8. 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. PMID: 15464952.

9. Zuo, X. and Speicher, D.W. 2004. Microscale solution isoelectrofocusing: A sample prefractionation method for comprehensive proteome analysis. Methods in Mol. Biol. 244:361-375. PMID: 14970573.

10. 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. PMID: 16052620.

11. 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. PMID: 16052622.

12. 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. PMID: 16000077.

13. 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. Nat. Biotechnol. 24:333-338. PMID: 16525410.

14. Hoffman, S.A., Joo, W-A., Echan, L.A., and Speicher, D.W. 2008. Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma. J. Chromatogr. B. 849:43-52. PMID: 17140865.

15. 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. PMID: 17494944.

16. 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. PMID: 17673662.

17. 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. PMID: 17977835.

18. 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. 9:768-782. PMID: 19132682. PMCID: PMC2667946.

19. 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. 419:669-679. PMID: 19140803. PMCID: PMC2770719.

20. Bell, A.W., Deutsch, E.W., Au, C.E., Kearney, R.E., Beavis, R., Sechi, S., Nilsson, T., Bergeron, J.J.; HUPO Test Sample Working Group, Beardslee, T.A., Chappell, T., Meredith, G., Sheffield, P., Gray, P., Hajivandi, M., Pope, M., Predki, P., Kullolli, M., Hincapie, M., Hancock, W.S., Jia, W., Song, L., Li, L., Wei, J., Yang, B., Wang, J., Ying, W., Zhang, Y., Cai, Y., Qian, X., He, F., Meyer, H.E., Stephan, C., Eisenacher, M., Marcus, K., Langenfeld, E., May, C., Carr, S.A., Ahmad, R., Zhu, W., Smith, J.W., Hanash, S.M., Struthers, J.J., Wang, H., Zhang, Q., An, Y., Goldman, R., Carlsohn, E., van der Post, S., Hung, K.E., Sarracino, D.A., Parker, K., Krastins, B., Kucherlapati, R., Bourassa, S., Poirier, G.G., Kapp, E., Patsiouras, H., Moritz, R., Simpson, R., Houle, B., Laboissiere, S., Metalnikov, P., Nguyen, V., Pawson, T., Wong, C.C., Cociorva, D., Yates III, J.R., Ellison, M.J., Lopez-Campistrous, A., Semchuk, P., Wang, Y., Ping, P., Elia, G., Dunn, M.J., Wynne, K., Walker, A.K., Strahler, J.R., Andrews, P.C., Hood, B.L., Bigbee, W.L., Conrads, T.P., Smith, D., Borchers, C.H., Lajoie, G.A., Bendall, S.C., Speicher, K.D., Speicher, D.W., Fujimoto, M., Nakamura, K., Paik, Y.K., Cho, S.Y., Kwon, M.S., Lee, H.J., Jeong, S.K., Chung, A.S., Miller, C.A., Grimm, R., Williams, K., Dorschel, C., Falkner, J.A., Martens, L., Vizcaíno, J.A. 2009. A HUPO test sample study reveals common problems in mass spectrometry-based proteomics. Nat. Methods. 6:423-430. PMID: 19448641. PMCID: PMC2785450.

21. Wang, H., Chang-Wong, T., Tang, H-Y., and Speicher, D.W. 2010. Comparison of extensive protein fractionation and repetitive LC-MS/MS analyses on depth of analysis for complex proteomes. J. Proteome Res. 9:1032-40. PMID: 20014860. PMCID: PMC2870931.

22. Li, D., Harper, S.L., Tang, H.Y., Maksimova, Y., Gallagher, P.G., and Speicher, D.W. 2010. A comprehensive model of the spectrin divalent tetramer binding region deduced using homology modeling and chemical cross-linking of a mini-spectrin. J. Biol. Chem. 285:29535-29545. PMID: 20610390. PMCID: PMC2937985.

23. Han, M-J., Wang, H., Beer, L., Tang, H.-Y., Herlyn, M., and Speicher, D.W. 2010. A systems biology analysis of metastatic melanoma using in-depth three-dimensional protein profiling. Proteomics 10:4450-4462. PMID: 21136598. PMCID: PMC307844.

24. Fong, K.P., Barry, C., Tran, A.N., Traxler, E.A., Wannemacher, K.M., Tang, H-Y., Speicher, K.D., Blair, I.A., Speicher, D.W., Grosser, T., and Brass, L.W. 2011. Deciphering the human platelet sheddome. Blood. 117:e15-26. PMID: 20962327. PMCID: PMC3037762.

25. Beer, L.A., Tang, H.-Y., Sriswasdi, S., Barnhart, K.T., and Speicher, D.W. 2011. Systematic discovery of ectopic pregnancy serum biomarkers using 3-D protein profiling coupled with label-free quantitation. J Proteome Research. 10:1126-1138. PMID: 21142075. PMCID: PMC3048922.

26. Chatterjee, S., Feinstein, S.I., Dodia, C., Nguyen, S., Lien, Y.-C., Sorokina, E., Debolt, K., Speicher, D., and Fisher, A.B. 2011. Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium. J Biol Chem. 286:11696-11706. PMID: 21262967. PMCID: PMC3064221.

27. Krieger, C.C., An, X., Tang, H.-Y., Mohandas, N., Speicher, D.W., and Discher, D.E. 2011. Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells. Proc. Natl. Acad. Sci. USA. 108:8269-8274. PMID: 21527722. PMCID: PMC3100976.

28. Yi, C., Troutman, S., Fera, D., Stemmer-Rachamimov, A., Avila, J.L., Christian, N., Persson, N.L., Shimono, A., Speicher, D.W., Marmorstein, R., Holmgren, L., and Kissil, J.L. 2011. A tight junction-associated Merlin-angiomotin complex mediates Merlin's regulation of mitogenic signaling and tumor suppressive functions. Cancer Cell. 19:527-540. PMID: 21481793. PMCID: PMC3075552.

29. Tang, H.-Y., Beer, L.A., and Speicher, D.W. 2011. In-depth analysis of a plasma or serum proteome using a 4D protein profiling method. Methods Mol Biol. 728:47-67. PMID: 21468940. PMCID: PMC3157844.

30. Beer, L.A., Tang, H.-Y., Barnhart, K.T., and Speicher, D.W. 2011. Plasma biomarker discovery using 3D protein profiling coupled with label-free quantitation. Methods Mol Biol. 728:3-27. PMID: 21468938. PMCID: PMC3157887.

31. Tang, H.-Y., Beer, L.A., Barnhart, K.T., and Speicher, D.W. 2011. Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring. J. Proteome Res. 10:4005-17. PMID: 21726088. PMCID: PMC3166403.

32. Wang, H., Tang, H.-Y., Tan, G.C., and Speicher, D.W. 2011. A data analysis strategy for maximizing high-confidence protein identifications in complex proteomes such as human tumor secretomes and human sera. J. Proteome Res. 10:4993-5005.  PMID: 21955121. PMCID: PMC3221390.

33. Yuan, H., Rossetto, D., Mellet, H., Dang, W., Madhusudan, S., Hodawadekar, S., Ding, E.C., Speicher, K., Abshiru, N., Perry, R, Speicher, D.W., Thibault, P., Verreault, A., Berger, S.L., Sternglanz, R., McMahon, S.B., Côté, J., and Marmorstein, R. 2011. MYST protein acetyltransferase activity requires active site lysine autoacetylation. EMBO J. 31:58-70. PMID: 22020126. PMCID: PMC3252582.

34. Tang, H.-Y., Beer, L., Chang-Wong, T., Hammond, R., Gimotty, P., Coukos, G., and Speicher, D.W. 2012. A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer. J Proteome Res. 11:678-91. PMID: 22032327. PMCID: PMC3272104.

35. Bhattacharjee, S., Stahelin, R.V., Speicher, K.D., Speicher, D.W., Haldar, K. 2012. Endoplasmic reticulum PI(3)P lipid binding targets malaria proteins to the host cell. Cell. 148:201-12. PMID:22265412. PMCID: PMC3268671.

36. Cao, Z., Tang, H.-Y., Wang, H., Liu, Q., and Speicher, D.W. 2012. Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes. J. Proteome Res. Apr 26. [Epub ahead of print]. PMID: 22536952. NIHMS374305.