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 PREPARATIONNote: 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 mostserum-containing media will innately contain cytokines. If testing serum-containing medium, it isrecommended 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 serumcontaining 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 10minutes. Save the supernatant. Transfer the supernatant into 1.5 ml Eppendorf tubes. Storesupernatant 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 willneed to be determined by each customer empirically based on researched literature and knowledgeof the particular samples.

How do I prepare cell or tissue lysate samples?Cell or tissue lysates for use with ELISA kits can be prepared usingmost conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply acompatible lysis buffer in many of our kits but other general low-salt (700 mM) lysis buffers can beused with the following caveats:

1) Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionicdetergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such asCHAPS, or mild ionic detergents such as sodium deoxycholate will work.2) Use no more than 2% v/v total detergent3) Avoid the use of sodium azide4) 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 tohomogenization. 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 sampletypes. Your choice of method should be made following a brief search of the literature to see howsamples 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 10min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon aspossible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with anyimmunoassay. Next, determine the protein concentration of your lysates using a total protein assaynot inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume ofeach 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, wesuggest using 500 uL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust thisbased upon your results. Your target for total protein concentration of the homogenate should be atleast 1,000 ug/mL, but 2,000 ug/mL or more would be better."

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!https://www.abcam.com/abreviews

回复于 Nov 07 2012