Overview

  • Product name

    Mouse RAGE ELISA Kit
    See all RAGE kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Serum, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 15 pg/ml
  • Range

    13.72 pg/ml - 10000 pg/ml
  • Recovery

    87 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 75.89 67% - 82%
    Serum 102.9 91% - 110%
    Plasma 82.69 71% - 92%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse
  • Product overview

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

    This assay employs an antibody specific for mouse RAGE coated on a 96-well plate. Standards and samples are pipetted into the wells and RAGE present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-mouse RAGE 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 RAGE bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

  • 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
    160X HRP-Streptavidin Concentrate 1 x 200µl
    20X Wash Buffer Concentrate 1 x 25ml
    5X Assay Diluent 1 x 15ml
    Biotinylated anti-mouse RAGE (lyophilized) 2 vials
    RAGE Microplate (12 x 8 wells) 1 unit
    Recombinant Mouse RAGE Standard (lyophilized) 2 vials
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
  • Research areas

  • Function

    Mediates interactions of advanced glycosylation end products (AGE). These are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Acts as a mediator of both acute and chronic vascular inflammation in conditions such as atherosclerosis and in particular as a complication of diabetes. AGE/RAGE signaling plays an important role in regulating the production/expression of TNF-alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes. Interaction with S100A12 on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Interaction with S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53/TP53 signaling (By similarity). Receptor for amyloid beta peptide. Contributes to the translocation of amyloid-beta peptide (ABPP) across the cell membrane from the extracellular to the intracellular space in cortical neurons. ABPP-initiated RAGE signaling, especially stimulation of p38 mitogen-activated protein kinase (MAPK), has the capacity to drive a transport system delivering ABPP as a complex with RAGE to the intraneuronal space.
  • Tissue specificity

    Endothelial cells.
  • Sequence similarities

    Contains 2 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Cellular localization

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

    • Advanced glycosylation end product-specific receptor
    • Ager
    • DAMA 358M23.4
    • MGC2235
    • MGC22357
    • RAGE_HUMAN
    • Receptor for advanced glycation end products
    • Receptor for advanced glycosylation end products
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab100738 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

  • Standard curve of mouse RAGE with background signal subtracted (duplicates; +/- SD).

  • Mouse RAGE measured in in mouse tissue lysates (in RIPA buffer tested in the range of 0.1ug to 0.1 mg protein, quantity of RAGE shown as per mg of extracted protein ), duplicates +/- SD.

  • Mouse RAGE measured in biological fluids (dilution range neat - 1/10; duplicates +/- SD).

  • Representative standard curve using ab100738

Protocols

References

ab100738 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

Tha lab sent their data for the two lots that gave you low standard OD compared to the June assay.

October


Standard (pg/ml) OD
10,000 2.832
3,333 1.512
1,111 0.732
370.4 0.351
123.5 0.201
41.15 0.151
13.72 0.134
0 0.122


November


Standard (pg/ml) OD
10,000 2.912
3,333 1.575
1,111 0.741
370.4 0.359
123.5 0.208
41.15 0.156
13.72 0.138
0 0.127


So, I am not sure what happened. Two possibilities are degraded standard compared to the samples they tested, and procedural inconsistency. I have added below some advice on preparing the standard solution which may seem obvious, but I am sending it in the hope that it is helpful. Please let me know if you have any questions or if you continue to see low OD values for the standards.

Improving Inter-assay Variability

There are a number of factors which can introduce variability between experiments. Please take the following precautions to minimize day-to-day fluctuations in your overall signal.

1) In general, try to stick with the same equipment and conditions from experiment to experiment, including pipets, plate shaker, shaker speed, and water source.
2) Please ensure proper kit storage. 4° C is sufficient for 6 months or less. For longer term storage we recommend -20° C.
3) Once the protein standard is reconstituted, the stock solution may be stored frozen (preferably at -80°C) for 2 or 3 weeks; however, degradation may occur after long-term storage of reconstituted protein. We recommend using a new fresh tube of lyophilized standard for each new experiment.
4) Incubation times should be followed exactly. The incubation times for the HRP-streptavidin and TMB substrate are particularly critical; even a 5-minute time difference can introduce a noticeable change in OD. Also note that overnight incubation in some steps may cause higher overall signals and higher background.
5) Ensure correct dilution of reagent concentrates: HRP-streptavidin, detection antibody, diluents and buffers.
6) When preparing the HRP-streptavidin, mix the vial (Item G) well before pipetting, as the solution may become slightly separated during long term storage. Since the solution is highly concentrated, pipet at least 2 µl of solution from the vial (Item G) to avoid pipet error.
7) To obtain consistent standard curves, ensure correct preparation of protein standard. Always centrifuge the vial before adding diluent. After the diluent is added, we then recommend inverting and flicking the tube a few times, and then spin it down again; repeat this procedure 3-4 times. This is a technique we find very effective for thoroughly mixing the standard without too much mechanical force.
8) Check pipet calibration and accurate volume dispensing; also try to minimize bubbles in the wells.
9) Ensure consistent shaking. We recommend 1-2 cycles per second on a rotary shaker or rocker.
10) Avoid temperature fluctuations.

Please note the following precautions to ensure a stronger OD response from your standard:

1. When preparing your standards, it is very critical to briefly centrifuge the vial first. The powder may drop off from the cap when opening it if you do not centrifuge. Be sure to dissolve the powder thoroughly when reconstituting. After adding Assay Diluent to the vial, we recommend inverting the tube a few times, then flick the tube a few times, and then spin it down; repeat this procedure 3-4 times. This is a technique we find very effective for thoroughly mixing the standard without too much mechanical force.
2. Avoid vortexing the standard during reconstitution, as this may destabilize the protein.
3. Once your standard has been reconstituted, it should be used right away or else frozen for later use.
4. Keep the standard dilutions on ice while during preparation, but the ELISA procedure should be done at room temperature.
5. Be sure to discard the working standard dilutions after use – they do not store well.

And keep in mind that that we provide 2 vials of lyophilized standard in the kit, to minimize storage issues. The unopened vials will be stable at 4 degrees for several months.

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Question
Answer

Thank you for contacting us.

The recommended dilution for testing normal mouse serum and plasma with this kit is 2-fold.

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.  This ELISA kit has not been validated for use on tissue samples, but may be suitable for that purpose.  Theoretically, any low-salt lysis buffer that meets the following criteria should be compatible with this kit: 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 

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