The Human AP-1 ELISA Kit is a powerful tool for researchers looking to accurately measure levels of AP-1 (Activator Protein 1) in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit offers reliable and reproducible results, making it a valuable asset for a wide range of research applications.AP-1 is a transcription factor complex that regulates gene expression and plays a crucial role in various biological processes, including cell proliferation, differentiation, and apoptosis. Dysregulation of AP-1 has been implicated in numerous diseases, including cancer, inflammatory disorders, and neurodegenerative conditions, making it a key target for studying these pathologies and developing therapeutic interventions.
Whether investigating the molecular mechanisms underlying disease progression or exploring potential therapeutic strategies, the Human AP-1 ELISA Kit provides researchers with a precise and efficient tool for detecting and quantifying AP-1 levels, paving the way for advancements in the field of biomedical research.
This immunoassay kit allows for the in vitro quantitative determination of Human JUN concentrations in serum plasma and other biological fluids.
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
Storage:
4°C for 6 months
Note:
For Research Use Only
Recovery:
Matrices listed below were spiked with certain level of Human JUN and the recovery rates were calculated by comparing the measured value to the expected amount of Human JUN in samples.
Matrix
Recovery range(%)
Average(%)
serum(n=5)
86-105
97
EDTA plasma(n=5)
92-105
99
UFH plasma(n=5)
86-102
91
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human JUN and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample
1:2
1:4
1:8
serum(n=5)
87-100%
87-105%
93-104%
EDTA plasma(n=5)
93-101%
85-96%
84-101%
UFH plasma(n=5)
80-95%
80-94%
91-96%
CV(%):
Intra-Assay: CV<8% Inter-Assay: CV<10%
Component
Quantity
Storage
ELISA Microplate (Dismountable)
8×12 strips
4°C for 6 months
Lyophilized Standard
2
4°C/-20°C
Sample/Standard Dilution Buffer
20ml
4°C
Biotin-labeled Antibody(Concentrated)
120ul
4°C (Protect from light)
Antibody Dilution Buffer
10ml
4°C
HRP-Streptavidin Conjugate(SABC)
120ul
4°C (Protect from light)
SABC Dilution Buffer
10ml
4°C
TMB Substrate
10ml
4°C (Protect from light)
Stop Solution
10ml
4°C
Wash Buffer(25X)
30ml
4°C
Plate Sealer
5
-
Other materials and equipment required:
Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Transcription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG]TCA-3'. Promotes activity of NR5A1 when phosphorylated by HIPK3 leading to increased steroidogenic gene expression upon cAMP signaling pathway stimulation. Involved in activated KRAS-mediated transcriptional activation of USP28 in colorectal cancer (CRC) cells (PubMed:24623306). Binds to the USP28 promoter in colorectal cancer (CRC) cells (PubMed:24623306).
NCBI Summary:
This gene is the putative transforming gene of avian sarcoma virus 17. It encodes a protein which is highly similar to the viral protein, and which interacts directly with specific target DNA sequences to regulate gene expression. This gene is intronless and is mapped to 1p32-p31, a chromosomal region involved in both translocations and deletions in human malignancies. [provided by RefSeq, Jul 2008]
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
Step
Protocol
1.
Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.
Aliquot 0.1ml standard solutions into the standard wells.
3.
Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.
Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.
Seal the plate with a cover and incubate at 37 °C for 90 min.
6.
Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.
Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.
Seal the plate with a cover and incubate at 37°C for 60 min.
9.
Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.
Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.
Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.
Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.
Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.
Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
