Overview

  • Product name

    Human Fas ELISA Kit (Apo-1)
    See all Fas kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Serum, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 5 pg/ml
  • Range

    2.74 pg/ml - 2000 pg/ml
  • Recovery

    > 85 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 91.24 82% - 105%
    Serum 84.46 78% - 102%
    Plasma 86.37 80% - 104%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s Fas Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human Fas in serum, plasma, and cell culture supernatants.


    This assay employs an antibody specific for Human Fas coated on a 96-well plate. Standards and samples are pipetted into the wells and Fas present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human Fas antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Fas bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    Get results in 90 minutes with Human FASLG Receptor ELISA Kit (CD95) (ab183360) from our SimpleStep ELISA® range.

  • Notes

    Optimization may be required with urine samples

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    20X Wash Buffer Concentrate 1 x 25ml
    5X Assay Diluent B 1 x 15ml
    640X HRP-Streptavidin Concentrate 1 x 200µl
    Assay Diluent A 1 x 30ml
    Biotinylated anti-Human Fas (lyophilized) 2 vials
    Fas Microplate (12 strips x 8 wells) 1 unit
    Recombinant Human Fas Standard (lyophilized) 2 vials
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
  • Research areas

  • Function

    Receptor for TNFSF6/FASLG. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. FAS-mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both. The secreted isoforms 2 to 6 block apoptosis (in vitro).
  • Tissue specificity

    Isoform 1 and isoform 6 are expressed at equal levels in resting peripheral blood mononuclear cells. After activation there is an increase in isoform 1 and decrease in the levels of isoform 6.
  • Involvement in disease

    Defects in FAS are the cause of autoimmune lymphoproliferative syndrome type 1A (ALPS1A) [MIM:601859]; also known as Canale-Smith syndrome (CSS). ALPS is a childhood syndrome involving hemolytic anemia and thrombocytopenia with massive lymphadenopathy and splenomegaly.
  • Sequence similarities

    Contains 1 death domain.
    Contains 3 TNFR-Cys repeats.
  • Domain

    Contains a death domain involved in the binding of FADD, and maybe to other cytosolic adapter proteins.
  • Cellular localization

    Secreted and Cell membrane.
  • Information by UniProt
  • Alternative names

    • ALPS 1A
    • ALPS1A
    • APO 1
    • Apo 1 antigen
    • APO 1 cell surface antigen
    • Apo-1 antigen
    • APO1
    • Apo1 antigen
    • APO1 cell surface antigen
    • Apoptosis antigen 1
    • Apoptosis mediating surface antigen FAS
    • Apoptosis-mediating surface antigen FAS
    • APT 1
    • APT1
    • CD 95
    • CD 95 antigen
    • CD95
    • CD95 antigen
    • Delta Fas
    • Delta Fas/APO 1/CD95
    • Delta Fas/APO1/CD95
    • Fas
    • Fas (TNF receptor superfamily, member 6)
    • FAS 1
    • FAS 827dupA
    • Fas AMA
    • FAS Antigen
    • Fas cell surface death receptor
    • FAS1
    • FASLG receptor
    • FASTM
    • sFAS
    • Surface antigen APO1
    • TNF receptor superfamily, member 6
    • TNFRSF 6
    • TNFRSF6
    • TNR6_HUMAN
    • Tumor necrosis factor receptor superfamily member 6
    see all
  • Database links

Associated products

Applications

Our Abpromise guarantee covers the use of ab100513 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Representative Standard Curve using ab100513.

  • Representative Standard Curve using ab100513.

Protocols

References

ab100513 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

Thank you for your enquiry.

Regarding the standard curve for ab100513 Fas Human ELISA Kit , I can confirm that the readings would be individual based on each individual experiment. A standard curve would need to be run each time.

There are some example standard curves available on the online datasheet and also in the protocols booklet (link at the bottom of the datasheet:
https://www.abcam.com/index.html?datasheet=100513 (or use the following: https://www.abcam.com/index.html?datasheet=100513).

We recommend a 2 - 5 fold dilution for normal human serum/plasma samples for use with this Human Fas ELISA kit.

For saliva samples we would recommend a minimal dilution, as cytokine concentrations are typically much less in this sample type. Thus, a 1 - 2 fold dilution of saliva samples would be a good starting point. As always, levels of the target protein may vary between different specimens, so, optimal dilution factors for each sample must be determined by the investigator.

We recommend using Assay Diluent B for saliva samples. Assay Diluent B should be used for any sample type other than serum/plasma.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your enquiry.

Please find some useful information about the sample preparation below. It is important to note that the optimal sample preparation methods will need to be determined by the researcher.

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?



We recommend preparing serum-free or low-serum medium samples, as serum tends to contain cytokines which may produce significant background signals. If it is necessary to test serum-containing medium, we recommend also running an uncultured media blank to assess baseline signals. This baseline can then be subtracted from the cultured media sample data.

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.

*The optimal number of seeded cells varies from one cell type to another and may need to be empirically determined.

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 for use with this ELISA kit can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. You may also use your own lysis buffer, such as RIPA or other formulations optimized for immunoprecipitation. Please note the following guidelines on lysis buffer composition:

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

We strongly recommend adding a 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 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 best method for all 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 anyimmunoassay. 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 μL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target total protein concentration of the homogenate should be at least 1,000 μg/mL, but 2,000 μg/mL or more would be better.

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for contacting us and reporting the problems you have observed with the CD95 Human ELISA Kit (ab100513).

Fas is an alternative name for CD95, could you check for me if the 96 well plates were labeled with "Fas" or "Fas ligand"

Please could you check this for me and let me know. I look forward to receiving your reply.

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Answer

Thank you for your reply.

I can confirm that these kits will detect both soluble and membrane bound Fas and fas ligand.

In order to prevent membrane bound protein to interfere with your results, I suggest to spin down the saliva samples to get rid of all cells while taking care not to generate cell debris.

Plasma is is in generally cell free and therefore should not contain any membrane bound proteins.

I am sorry that I could not be of more help and hope this information is nevertheless helpful.

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Answer

Thank you for your enquiry.

I have reviewed the details on the datasheets for these kits. For the Fas ligand kits:

ab45892 Fas Ligand Human ELISA Kit
ab100515 Fas Ligand Human ELISA Kit

Sensitivities are different:ab100515 < 2 pg/ml, ab45892 < 12 pg/ml

Detection ranges are different:ab10515 1.37 pg/ml - 1000 pg/ml,ab45892 62.5 pg/ml - 2000 pg/ml

For the CD95 kits:
ab45922 CD95 Human ELISA Kit
ab100513 CD95 Human ELISA Kit

Again, sensitivities are different: ab100513 < 5 pg/ml, ab45922 < 47 pg/ml
Detection ranges are different:ab45922 93.75 pg/ml - 3000 pg/ml, ab100513 2.74 pg/ml - 2000 pg/ml

These are the main differences you may need to take into consideration. However, I can recommend to review the datasheets fullyfor further information and otherdifferences.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your inquiry.

These ELISA kits are from two different sources but they are similar.

I recommend todetermine which is the best foryour needs from the datasheet and protocol information. Please take not of the different ranges these kits are suitable for. I suggest to choose the kits that are most suitable for the amounts of Fas ligand and CD95 you are expecting in your samples.

I believe that all these kits are suitable for un-bound (free) Fas ligand and CD95 since for ELISA the cells are lysed and ELISA is not a cell based assay.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

The best kit will depend on the amount of FAS (CD95) that you are expecting to detect in your samples. The kit ab100513 produces a standard curve that is linear for lower concentrations of FAS (CD95) than ab119571. Both of these kits will be guaranteed for use on human serum samples, but have not been tested on saliva samples. In theory, they should work for saliva, but the sample dilution will need to be optimized.

I hope this helps, please let me know if you need any additional information or assistance.

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