The Rat ACTN4 (Alpha-Actinin-4) ELISA Kit is a reliable tool for detecting and quantifying levels of ACTN4 in rat samples. This kit offers high sensitivity and specificity, providing accurate and reproducible results for a variety of research applications.ACTN4 is a key protein involved in cellular structure and function, playing a crucial role in cell adhesion and migration. Dysregulation of ACTN4 has been linked to various diseases, including cancer, kidney disorders, and cardiovascular diseases.
Therefore, measuring ACTN4 levels can provide valuable insights into disease progression and potential therapeutic interventions.With its superior performance and ease of use, the Rat ACTN4 ELISA Kit is an essential tool for researchers studying the role of ACTN4 in health and disease. Order now to accelerate your research and uncover new insights into cellular biology and disease mechanisms.
Product Name:
Rat Alpha-actinin-4 (Actn4) ELISA Kit
SKU:
RTEB0985
Size:
96T
Target:
Rat Alpha-actinin-4 (Actn4)
Synonyms:
Non-muscle alpha-actinin 4
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
78-5000pg/mL
Sensitivity:
39.14pg/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Probably involved in vesicular trafficking via its association with the CART complex. The CART complex is necessary for efficient transferrin receptor recycling but not for EGFR degradation. Involved in tight junction assembly in epithelial cells probably through interaction with MICALL2. Links MICALL2 to the actin cytoskeleton and recruits it to the tight junctions. May also function as a transcriptional coactivator, stimulating transcription mediated by the nuclear hormone receptors PPARG and RARA.
Uniprot:
Q9QXQ0
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Alpha-actinin-4
Sub Unit:
Homodimer; antiparallel (By similarity). Interacts with BAIAP1 (By similarity). Interacts with MICALL2 (preferentially in opened conformation); stimulated by RAB13 activation (By similarity). Identified in a IGF2BP1-dependent mRNP granule complex containing untranslated mRNAs. Component of the CART complex, at least composed of ACTN4, HGS/HRS, MYO5B and TRIM3. Binds TRIM3 at the N-terminus. Interacts with PDLIM2. Identified in a complex with CASK, IQGAP1, MAGI2, NPHS1, SPTAN1 and SPTBN1. Interacts with PPARG and RARA (By similarity). Binds to VCL; this interaction triggers VCL conformational changes.
Research Area:
Epigenetics
Subcellular Location:
Nucleus Cytoplasm Cell junction Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Colocalizes with actin stress fibers.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
ACTN4: F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Probably involved in vesicular trafficking via its association with the CART complex. The CART complex is necessary for efficient transferrin receptor recycling but not for EGFR degradation. Defects in ACTN4 are the cause of focal segmental glomerulosclerosis type 1 (FSGS1). A renal pathology defined by the presence of segmental sclerosis in glomeruli and resulting in proteinuria, reduced glomerular filtration rate and edema. Renal insufficiency often progresses to end-stage renal disease, a highly morbid state requiring either dialysis therapy or kidney transplantation. Belongs to the alpha-actinin family.Protein type: Motility/polarity/chemotaxis; CytoskeletalCellular Component: cortical cytoskeleton; extracellular space; neuron projection; focal adhesion; protein complex; pseudopodium; intercellular junction; Z disc; cytosol; ribonucleoprotein complex; perinuclear region of cytoplasm; cytoplasm; stress fiber; nucleusMolecular Function: actin filament binding; protein binding; protein homodimerization activity; ligand-dependent nuclear receptor transcription coactivator activity; retinoic acid receptor binding; nucleoside binding; protein complex binding; protein N-terminus binding; calcium ion binding; nuclear hormone receptor bindingBiological Process: retinoic acid receptor signaling pathway; regulation of apoptosis; actin filament bundle formation; protein transport; response to hypoxia; negative regulation of cell motility; positive regulation of cell motility; positive regulation of pinocytosis
UniProt Protein Details:
NCBI Summary:
alpha-actinin cytoskeletal protein that anchors various actin-containing microfilaments to cellular structures; anchors V myosins to cellular structures via the RING finger-containing protein BERP [RGD, Feb 2006]
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.
