The Rat Profilin-1 (PFN1) ELISA Kit is specifically designed for the precise measurement of profilin-1 levels in rat serum, plasma, and cell culture supernatants. This ELISA kit offers excellent sensitivity and specificity, ensuring accurate and consistent results for a variety of research purposes.Profilin-1 is a key protein involved in regulating actin polymerization, cell migration, and cell signaling pathways.
Its dysfunction has been linked to various pathological conditions, including cancer, neurodegenerative diseases, and autoimmune disorders. Thus, the Rat Profilin-1 (PFN1) ELISA Kit serves as a valuable tool for investigating the role of profilin-1 in these diseases and potential therapeutic interventions.
Product Name:
Rat Profilin-1 (Pfn1) ELISA Kit
SKU:
RTEB1380
Size:
96T
Target:
Rat Profilin-1 (Pfn1)
Synonyms:
Profilin I
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.312-20 ng/mL
Sensitivity:
0.08 ng/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
Binds to actin and affects the structure of the cytoskeleton. At high concentrations, profilin prevents the polymerization of actin, whereas it enhances it at low concentrations. By binding to PIP2, it inhibits the formation of IP3 and DG. Inhibits androgen receptor (AR) and HTT aggregation and binding of G-actin is essential for its inhibition of AR.
Uniprot:
P62963
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Profilin-1
Sub Unit:
Interacts with VASP. Occurs in many kinds of cells as a complex with monomeric actin in a 1:1 ratio. Found in a complex with XPO6, Ran, ACTB and PFN1 (By similarity). Interacts with HTT.
Research Area:
Cancer
Subcellular Location:
Cytoplasm Cytoskeleton
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
profilin 1: a ubiquitous actin monomer-binding protein belonging to the profilin family. At high concentrations, profilin 1 prevents the polymerization of actin, whereas it enhances it at low concentrations. By binding to PIP2, it inhibits the formation of IP3 and DG. Deletion of this gene is associated with Miller-Dieker syndrome. Protein type: Motility/polarity/chemotaxis; Cytoskeletal; Actin-bindingCellular Component: neuron projection; focal adhesion; cytoskeleton; membrane; cytoplasm; synapse; nucleusMolecular Function: protein binding; phosphatidylinositol-4,5-bisphosphate binding; Rho GTPase binding; adenyl-nucleotide exchange factor activity; actin binding; receptor bindingBiological Process: positive regulation of DNA metabolic process; negative regulation of actin filament bundle formation; negative regulation of stress fiber formation; negative regulation of actin filament polymerization; positive regulation of viral transcription; regulation of transcription from RNA polymerase II promoter; response to organic substance; positive regulation of actin filament polymerization; positive regulation of ATPase activity; actin polymerization and/or depolymerization; positive regulation of stress fiber formation; neural tube closure; positive regulation of transcription from RNA polymerase II promoter
UniProt Protein Details:
NCBI Summary:
regulates actin polymerization; may reorganize the actin cytoskeleton; may be involved in the pathogenesis of glomerulonephritis [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.
