Rat Sterol regulatory element-binding protein 2 (Srebf2) ELISA Kit
The Rat Sterol Regulatory Element-Binding Protein 2 (SREBF2) ELISA Kit is a powerful tool for accurately measuring the levels of SREBF2 in rat samples, including serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides reliable and reproducible results for a variety of research applications.SREBF2 is a key transcription factor that plays a crucial role in regulating lipid metabolism and cholesterol biosynthesis. Dysregulation of SREBF2 has been linked to various metabolic disorders, making it a valuable biomarker for studying these conditions and potential therapeutic targets.
This ELISA kit offers researchers a convenient and reliable way to quantify SREBF2 levels, allowing for deeper insights into its function and implications in metabolic diseases. With its user-friendly format and high-performance characteristics, the Rat SREBF2 ELISA Kit is an essential tool for any laboratory conducting research in the field of lipid metabolism and cardiovascular health.
Product Name:
Rat Sterol regulatory element-binding protein 2 (Srebf2) ELISA Kit
SKU:
RTEB0768
Size:
96T
Target:
Rat Sterol regulatory element-binding protein 2 (Srebf2)
Transcriptional activator required for lipid homeostasis. Regulates transcription of the LDL receptor gene as well as the cholesterol and to a lesser degree the fatty acid synthesis pathway (By similarity). Binds the sterol regulatory element 1 (SRE-1) (5'-ATCACCCCAC-3') found in the flanking region of the LDRL and HMG-CoA synthase genes.
Uniprot:
Q3T1I5
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Sterol regulatory element-binding protein 2
Sub Unit:
Forms a tight complex with SCAP in the ER membrane. Efficient DNA binding of the soluble transcription factor fragment requires dimerization with another bHLH protein. Interacts with LMNA. Component of SCAP/SREBP complex composed of SREBF2, SCAP and RNF139; the complex hampers the interaction between SCAP and SEC24B, thereby reducing SREBF2 proteolytic processing. Interacts (via C-terminal domain) with RNF139.
Research Area:
Cardiovascular
Subcellular Location:
Processed sterol regulatory element-binding protein 2 Nucleus
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
SREBP-2: Transcriptional activator required for lipid homeostasis. Regulates transcription of the LDL receptor gene as well as the cholesterol and to a lesser degree the fatty acid synthesis pathway. Binds the sterol regulatory element 1 (SRE-1) (5'-ATCACCCCAC-3') found in the flanking region of the LDRL and HMG-CoA synthase genes. Belongs to the SREBP family.Protein type: Transcription factor; Membrane protein, multi-pass; Membrane protein, integral; DNA-bindingChromosomal Location of Human Ortholog: 22q13Cellular Component: Golgi membrane; nucleoplasm; endoplasmic reticulum membrane; endoplasmic reticulum; cytoplasm; nucleolus; cytosol; nucleusMolecular Function: protein dimerization activity; protein C-terminus binding; protein binding; C-8 sterol isomerase activityBiological Process: cholesterol metabolic process; transcription, DNA-dependent; response to low density lipoprotein stimulus; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription from RNA polymerase II promoter; lipid metabolic process; cellular lipid metabolic process
UniProt Protein Details:
NCBI Summary:
This gene encodes a member of the a ubiquitously expressed transcription factor that controls cholesterol homeostasis by regulating transcription of sterol-regulated genes. The encoded protein contains a basic helix-loop-helix-leucine zipper (bHLH-Zip) domain and binds the sterol regulatory element 1 motif. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Jul 2013]
sterol regulatory element binding transcription factor 2
NCBI Official Symbol:
SREBF2
NCBI Official Synonym Symbols:
SREBP2; bHLHd2; SREBP-2
NCBI Protein Information:
sterol regulatory element-binding protein 2; class D basic helix-loop-helix protein 2; sterol regulatory element-binding transcription factor 2
UniProt Protein Name:
Sterol regulatory element-binding protein 2
UniProt Synonym Protein Names:
Class D basic helix-loop-helix protein 2; bHLHd2; Sterol regulatory element-binding transcription factor 2
Protein Family:
UniProt Gene Name:
SREBF2
UniProt Entry Name:
SRBP2_HUMAN
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
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
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.
