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Dear Sir or Madam,
I would be interested in the TGF beta 1 Human ELISA Kit - 1 x 96 Well Plate (ab100647). I will have to measure tgf concentration in human serum samples. Could you please provide information about the serum preparation specific for tgf analysis? How much time should I wait from blood sample collection before centrifugation for serum separation? At which speed?
Thanks in advance for your reply.
Asked on Feb 24 2012
Thank yhou for your enquiry.
I have copied some information belowproviding tips for sample preparation when using this kit, which includes human serum prepration.
I hope this will be helpful to you. if you have any further questions, please do not hesitate to contact me.
TIPS FOR SAMPLE PREPARATION
How do I make conditioned media for a sample?
For conditioned medium, it is better to prepare serum free or low serum medium since some sera also contains cytokines. At day 0, seed 1 million cells in 100 mm tissue culture plate with complete medium. At day 3, remove medium and replace medium with 6-8 ml of low serum medium (e.g. medium containing 0.2% calf serum). At day 5, medium is collected into 15 ml tube. Spin at 2,000 rpm in centrifuge at 4 C for 10 minutes. Save the supernatant. Transfer the supernatant into 1.5 ml eppendorf tubes. Store supernatant at -80oC until experiment. The sample can be stored at this way for at least one year.
Collect blood into EDTA or Sodium heparin tube
Spin 10 minutes at 3000 rpm
Aliquot into small tubes and store at -80oC until use.
Collect blood into tube without additive
Keep at room temperature for 20 minutes
Spin 10 minutes at 3000 rpm
Aliquot into small tubes and store at –80oC until use.
How do I prepare urine samples?
Collect samples without adding stabilizers. Spin down the samples hard (eg. 10000 x g for 1 min or 5000 x g for 2 min). Aliquot, quick freeze in dry ice/methanol bath, and store at -80oC until use.
How do I prepare cell or tissue lysates for use on the cytokine arrays?
The cell or tissue lysates for use with this kit can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply a compatible lysis buffer in most of our kits. Other general low-salt lysis buffers can be used with the following caveats:
1) Avoid using 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 reducing agents, such as dithiothreitol or mercaptoethanols
In general, we strongly recommend that you add 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, freeze-thaw, 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, spin down the lysates to remove cell/tissue debris (5 min @ 10000 x g or 10 min @ 5000 x g) and save the supernatant. Lysates should be frozen as soon as possible, and stored at -20oC (or -80oC, if possible). Spin them again before incubating with the antibody array. Determine the protein concentration of your lysates (for example the bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay.
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 1000 ug/mL, but 2000 ug/mL would be better.
Abcam Scientific Support
Answered on Feb 24 2012