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Is it possible to use VEGF ELISA kit (ab100663) for determining VEGF in bone tissue extracts as well?
Asked on Oct 16 2012
Thank you for your enquiry.
Unfortunately, we have been unable to find any references using bone tissue extracts with the VEGF ELISA kit ab100663 nor do we have tips for preparing this type of sample. You may wish to consider trying a further search for literature regarding preparation guidelines.
I have copied below some general tips on sample preparation, some of which may be helpful even for your bone samples so I can recommend to review this. I can also suggest to ensure not to use too much reducing or denaturing reagents when preparing the samples.
I hope this will be helpful. Please do not hesitate to let me know if you need further assistance.
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 plasma and serum samples?
1. Collect whole blood into an EDTA, Citrate or Sodium heparin tube
2. Centrifuge 10 minutes at 3,000 rpm
3. Aliquot into small tubes and store at -800C until use.
1. Collect whole blood into a tube without additives
2. Keep at room temperature for 20 minutes.
3. Centrifuge 10 minutes at 3,000 rpm.
4. Aliquot into small tubes and store at -800C until use.
How do I prepare urine samples?
1. Collect urine without adding stabilizers.
2. Centrifuge the samples hard (eg. 10,000 x g for 1 min or 5,000 x g for 2 min).
3. Aliquot, quick freeze in dry ice/methanol bath, and store at -800C until use.
How do I prepare cell or tissue lysate samples?
Cell or tissue lysates 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.
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 10min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as possible and stored at -20oC (or -80oC, 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.
Answered on Oct 16 2012