Any clue how resistant is it (boiling, long storage of the sample collection in the Laemli buffer)? This question I'm asking before the preparation of huge number of mammalian tissues collection in large amount I want to store for many years and share with other researchers. In general, the cost does not matter, the samples I want to store for western-blotting analyses.
We regularly use 5% BME in 1x loading buffer (with 10%glycerol). Very often I use 2x loading dye to directly lyse the samples (mostly invitro cell culture lysates), boil and store for few years at -80. With tissues, hope it works.
Dear Manoj - thank you for your fast reply!
This is exactly I do for the routine use. But the samples after year smell like a ..... not the BME, the loading is not perfect as well. I thought about DTT or TCEP and maybe speed-vac later to store them dry?
Tris[2-carboxyethyl] phosphine (TCEP) is a popular odorless reducing agent for protein applications.
Considerations for use of TCEP:
TCEP is generally very soluble in aqueous buffers at nearly any pH. Therefore, working concentrations and 10X stock solutions may be readily prepared in most aqueous buffers
TCEP is stable in aqueous, acidic, and basic solutions. When TCEP is dissolved directly in water, the resulting pH is approximately 2.5.
TCEP is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in PBS buffers, prepare the working solution immediately before use.
TCEP may be used as a substitute for DTT or 2-mercaptoethanol (2-ME) in sample loading buffer for SDS-PAGE; use a final concentration of 50 mM TCEP.
Because TCEP is charged in solution, it is not compatible for use in isoelectric focusing.
References:
Duchardt F. et al. (2009) A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency. J Biol Chem. 284, 36099-108.
Hou J. M. et al. (2008) Atpase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages. J. Bacteriol. 190, 4880-7.
Feng J. et al. (2008) Quantitative proteomic profiling of muscle type-dependent and age-dependent protein carbonylation in rat skeletal muscle mitochondria. J Gerontol A Biol Sci Med Sci. 63, 1137-52.
Chan C. T. et al. (2008) Molecular imaging of the efficacy of Heat Shock Protein 90 inhibitors in living subjects. Cancer Res. 68, 216-26.
Nossal N. G. et al. (2007) Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers. J. Biol. Chem. 282, 1098-108.
Follow this site for TCEP protocol
http://mullinslab.ucsf.edu/Protocols%20HTML/making_tcep_stocks.htm
Thank you Imran so much! Impressive! I will think about it for a long time, since PBS is my beloved buffer for protein preparation...
Any tips about TCEP range of concentration in SDS-PAGE loading buffer?
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