Mouse Adenomatous polyposis coli protein (Apc) ELISA Kit
The Mouse Adenomatous Polyposis Coli protein (APC) ELISA Kit is specifically designed for accurate detection of APC levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.APC is a key protein involved in regulating cell growth and division, with mutations in the APC gene being associated with various types of cancer, particularly colorectal cancer.
The APC protein also plays a critical role in the Wnt signaling pathway, which is important for embryonic development and tissue homeostasis.By accurately measuring APC levels, researchers can gain valuable insights into the role of APC in cancer development and progression, as well as its potential as a therapeutic target. The Mouse APC ELISA Kit provides a valuable tool for studying APC biology and its implications in cancer research.
Product Name:
Mouse Adenomatous polyposis coli protein (Apc) ELISA Kit
SKU:
MOEB0858
Size:
96T
Target:
Mouse Adenomatous polyposis coli protein (Apc)
Synonyms:
Protein APC
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.312-20ng/ml
Sensitivity:
0.166ng/mL
Intra CV:
6.8%
Inter CV:
9.3%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
111-121%
95-104%
101-111%
109-119%
EDTA Plasma(N=5)
95-103%
90-100%
100-100%
106-117%
Heparin Plasma(N=5)
100-110%
86-94%
88-98%
106-116%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
93
87-99
Plasma
95
89-101
Function:
Tumor suppressor. Promotes rapid degradation of CTNNB1 and participates in Wnt signaling as a negative regulator. APC activity is correlated with its phosphorylation state. Activates the GEF activity of SPATA13 and ARHGEF4. Plays a role in hepatocyte growth factor (HGF)-induced cell migration (By similarity). Required for MMP9 up-regulation via the JNK signaling pathway in colorectal tumor cells. Acts as a mediator of ERBB2-dependent stabilization of microtubules at the cell cortex. It is required for the localization of MACF1 to the cell membrane and this localization of MACF1 is critical for its function in microtubule stabilization.
Uniprot:
Q61315
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Adenomatous polyposis coli protein
Sub Unit:
Forms homooligomers and heterooligomers with APC2. Interacts with PDZ domains of DLG1 and DLG3. Associates with catenins. Binds axin. Interacts with MAPRE2 and MAPRE3 (via C-terminus). Found in a complex consisting of ARHGEF4, APC and CTNNB1. Interacts with ARHGEF4 (via N-terminus) (By similarity). Interacts with MAPRE1 (via C-terminus); probably required for APC targeting to the growing microtubule plus ends. Interacts with DIAPH1 and DIAPH2. Interacts with SCRIB; may mediate targeting to adherens junctions of epithelial cells. Interacts with SPATA13 (via N-terminus and SH3 domain). Interacts with ASAP1 (via SH3 domain) (By similarity). Found in a complex composed of MACF1, APC, AXIN1, CTNNB1 and GSK3B. Interacts at the cell membrane with AMER1 and AMER2 (via ARM repeats) (By similarity). Interacts with KHDRBS1.
Research Area:
Cancer
Subcellular Location:
Cell junction Adherens junction Cytoplasm Cytoskeleton Cell projection Lamellipodium Cell projection Ruffle membrane Cytoplasm Cell membrane Associated with the microtubule network at the growing distal tip of microtubules. Accumulates in the lamellipodium and ruffle membrane in response to hepatocyte growth factor (HGF) treatment. The MEMO1-RHOA-DIAPH1 signaling pathway controls localization of the phosphorylated form to the cell membrane.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Tumor suppressor. Promotes rapid degradation of CTNNB1 and participates in Wnt signaling as a negative regulator. APC activity is correlated with its phosphorylation state. Activates the GEF activity of SPATA13 and ARHGEF4. Plays a role in hepatocyte growth factor (HGF)-induced cell migration (). Required for MMP9 up-regulation via the JNK signaling pathway in colorectal tumor cells. Acts as a mediator of ERBB2-dependent stabilization of microtubules at the cell cortex. It is required for the localization of MACF1 to the cell membrane and this localization of MACF1 is critical for its function in microtubule stabilization ().
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
