Human VEGF ELISA Kit (ab100663)

Please note that ab100662 and ab100663 are similar kits that both measure levels of VEGF.

However ab100662 is best used for detecting VEGF in biological fluids such as cell culture supernatant, serum and plasma while ab100663 is best suited for use with cell and tissue extracts.

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?

For plasma:

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.

For serum:

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.

Thank you for your enquiry.

I am sorry to confirm we have not tested the effects of Urea containing lysis buffer on samples tested with ab100663 VEGF Human ELISA Kit. We would therefore not be able to confirm how well this would work. However, it is important to note that Urea is a Chaotropic agent at 6 - 8 M, meaning that it can denature proteins. Therefore this buffer formula may adversely affect the VEGF protein, causing it not to be recognized by the ELISA.

I am sorry we have no data to help answer your query on this occasion, however I hope the information is helpful. If you have any further questions, please do not hesitate to contact us.

Thank you for contacting us.

The concentration of the HRP-streptavidin reagent is considered proprietary information by the developer of the kit. I am sorry we could not be of more help.

Please do not hesitate to contact us if you have any other questions.

I am happy to hear that the new kits are working well for you! Please let me know if there is anything else I can do to help.

Thank you for contacting Abcam regarding our VEGF Human ELISA Kits.


The main difference between ab100663 and ab100662 is the sample types that can be analyzed. Ab100663 can be used to analyze cells and tissues, while ab100662 should be used to analyze serum, plasma, cell culture supernatants, and urine.


Please see the guidelines below for your sample preparation of cell or tissue lysates:


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. 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 protein 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 protein 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 -20C (or -80C, if possible). Spin them again before incubating with the antibody array. Determine the protein concentration of your lysates (using a Bradford-Lowry-type 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.

Thank you for contacting us. We unfortunately are not aware about the purpose of assaying VEGF in cell supernatant or in cell/tissue culture lysates. You may need to search literature to know more about this. The following publication will help to start. http://www.ncbi.nlm.nih.gov/pubmed/1380254 VEGF 165, 121 are the secreted protein and 189 and 206 are the membrane bound proteins so it will be interesting to know the total amount of VEGF in cell culture samples (supernatant + lysates) however it will depend on the type of experiment and results you will be expecting. Ab100662 is a best kit for detection of VEGF in cell/tissue culture lysates while ab100663 is best for VEGF detection in serum, plasma or cell culture supernatant. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your inquiry and your interest in our products. ab100663 has been specially designed and optimized for cell and tissue lysate samples. If you would like to use different type of sample, I would advise consideraing the serum, plasma, and cell culture supernatant Human VEGF ELISA Kit (ab100662) instead. If you need any further assistance in the future, please do not hesitate to contact me.

I sincerely apologize for the mix-up with the kit you had ordered.  I've put through a free of charge replacement of the Item D, 5X sample diluent.  We will ship this out to you once we receive the vial from the supplying lab. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research and again I appreciate your patience with this!