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HEpG2 cells, how to use this kit with this kind of samples?
Have liver cell lines ever been tested before?
Asked on Nov 07 2012
Vielen Dank für Ihren Anruf.
Leider ist es so, wie ich befürchtet habe, wir können leider keine Angabe zu diesen Kits mit HepG2 Zellen, bzw mit anderen Leberzelllinien machen. Grundsätzlich spricht aber nichts gegen die Verwendung dieses Kits mit Zellkulturlysaten.
"GENERAL TIPS FOR SAMPLE PREPARATION
Note: In case follow-up experiments are needed, it is strongly recommended to sub-aliquot all samples after preparation to minimize cytokine degradation from multiple freeze-thaw cycles.
How do I prepare conditioned media samples?
For testing conditioned medium, it is best to prepare serum-free or low serum medium as most
serum-containing media will innately contain cytokines. If testing serum-containing medium, it is
recommended to also run an uncultured media blank sample to assess the baseline responses.
1. On day 0, seed ˜1 million cells in 100 mm tissue culture plate with complete medium.*
2. On day 3, remove medium and replace medium with 6-8 ml of serum-free or low serum
containing medium (e.g. medium containing 0.2% calf serum).
3. On day 5, collect medium into 15 ml tube. Centrifuge at 2,000 rpm in centrifuge at 4ºC for 10
minutes. Save the supernatant. Transfer the supernatant into 1.5 ml Eppendorf tubes. Store
supernatant at -80ºC until experiment. Most samples can be stored this way for at least a year.
*Cell number may be lower or higher than this depending on the cell line so the optimal number will
need to be determined by each customer empirically based on researched literature and knowledge
of the particular samples.
How do I prepare cell or tissue lysate samples?
Cell or tissue lysates for use with ELISA kits can be prepared using
most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply a
compatible lysis buffer in many of our kits but other general low-salt (700 mM) lysis buffers can be
used with the following caveats:
1) Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionic
detergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such as
CHAPS, or mild ionic detergents such as sodium deoxycholate will work.
2) Use no more than 2% v/v total detergent
3) Avoid the use of sodium azide
4) Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols
Note: In general, any buffers used for immunoprecipitations, including RIPA buffer, should work.
We strongly recommend adding some type of protease inhibitor “cocktail” to the lysis buffer prior to
homogenization. Most general biochemical supply companies stock a wide variety of these products. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated (activated) forms of the protein.
Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw,
sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these.
There is no one “best method” for all sample types, but some are better than others for some sample
types. Your choice of method should be made following a brief search of the literature to see how
samples similar to yours have been prepared in previous investigations.
After homogenization, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10
min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as
possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any
immunoassay. Next, determine the protein concentration of your lysates using a total protein assay
not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of
each sample used to deliver the same amount of total protein for each assay.
Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.
Since different cells and tissues may contain different amounts of protein, as starting point, we
suggest using 500 uL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this
based upon your results. Your target for total protein concentration of the homogenate should be at
least 1,000 ug/mL, but 2,000 ug/mL or more would be better."
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Answered on Nov 07 2012