Rat Cell Biology ELISA Kits 1

Rat Breast cancer type 1 susceptibility protein homolog (Brca1) ELISA Kit

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SKU:
RTEB0519
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O54952
ELISA Type:
Sandwich
Reactivity:
Rat

Description

system_update_altDatasheetsystem_update_altMSDS
Product Name:Rat Breast cancer type 1 susceptibility protein homolog (Brca1) ELISA Kit
Product Code:RTEB0519
Alias:Breast cancer type 1 susceptibility protein homolog, Brca1, 6.3.2.-
Uniprot:O54952
Reactivity:Rat
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:BRCA1: the BRCA1-BARD1 heterodimer coordinates a diverse range of cellular pathways such as DNA damage repair, ubiquitination and transcriptional regulation to maintain genomic stability. Acts by mediating ubiquitin E3 ligase activity that is required for its tumor suppressor function. Plays a central role in DNA repair by facilitating cellular response to DNA repair. Required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. Involved in transcriptional regulation of P21 in response to DNA damage. Required for FANCD2 targeting to sites of DNA damage. May function as a transcriptional regulator. Inhibits lipid synthesis by binding to inactive phosphorylated ACC1 and preventing its dephosphorylation. Defects in BRCA1 are a cause of genetic susceptibility to breast cancer. Mutations in BRCA1 are thought to be responsible for more than 80% of inherited breast-ovarian cancer. Part of the BRCA1-associated genome surveillance complex (BASC), which contains BRCA1, MSH2, MSH6, MLH1, ATM, BLM, PMS2 and the RAD50-MRE11-NBN protein complex. This association may be a dynamic process changing throughout the cell cycle and within subnuclear domains. Interacts (via BRCT domains) with CTIP. Associates with RNA polymerase II holoenzyme. Interacts with SMC1 and COBRA1. Interacts (via BRCT domains) with BRIP1. Interacts with FANCD2 (ubiquitinated). Interacts with BAP1. Interacts with Artemis and claspin. Interacts with H2AFX (phosphorylated on S140). Interacts with CHK1. Interacts with BRCC3. Five human isoforms are produced by alternative splicing and alternative initiation. Isoform 1 and isoform 3 are widely expressed. Isoform 1 is largely nuclear, while isoforms 3 and 5 are cytoplasmic.Protein type: EC 6.3.2.-; Nuclear receptor co-regulator; Transcription, coactivator/corepressor; Tumor suppressor; Ubiquitin conjugating system; Ubiquitin ligaseChromosomal Location of Human Ortholog: 10q31Cellular Component: BRCA1-BARD1 complex; chromosome; condensed chromosome; condensed nuclear chromosome; cytoplasm; intracellular ribonucleoprotein complex; lateral element; mitochondrial matrix; nucleus; plasma membrane; protein complexMolecular Function: chromatin binding; damaged DNA binding; DNA binding; enzyme binding; protein binding; RNA binding; ubiquitin protein ligase binding; ubiquitin-protein transferase activity; zinc ion bindingBiological Process: brain development; cellular response to DNA damage stimulus; centrosome cycle; chordate embryonic development; chromosome segregation; DNA damage response, signal transduction resulting in induction of apoptosis; dosage compensation by inactivation of X chromosome; double-strand break repair; double-strand break repair via homologous recombination; fatty acid biosynthetic process; genetic imprinting; negative regulation of estrogen receptor signaling pathway; negative regulation of fatty acid biosynthetic process; negative regulation of histone acetylation; negative regulation of histone H3-K4 methylation; negative regulation of histone H3-K9 methylation; negative regulation of transcription, DNA-dependent; positive regulation of angiogenesis; positive regulation of DNA repair; positive regulation of histone acetylation; positive regulation of histone H3-K4 methylation; positive regulation of histone H3-K9 methylation; positive regulation of protein import into nucleus, translocation; positive regulation of protein ubiquitination; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-templated; postreplication repair; protein autoubiquitination; protein ubiquitination; regulation of cell proliferation; regulation of transcription from RNA polymerase II promoter; response to estradiol; response to estrogen; response to ionizing radiation; response to lipid; response to nutrient; response to organic substance; transcription, DNA-dependent
UniProt Protein Details:
NCBI Summary:involved in the DNA damage response and DNA repair [RGD, Feb 2006]
UniProt Code:O54952
NCBI GenInfo Identifier:6978573
NCBI Gene ID:497672
NCBI Accession:NP_036646.1
UniProt Secondary Accession:O54952,P97951
UniProt Related Accession:O54952
Molecular Weight:199,879 Da
NCBI Full Name:breast cancer type 1 susceptibility protein homolog
NCBI Synonym Full Names:BRCA1, DNA repair associated
NCBI Official Symbol:Brca1  
NCBI Official Synonym Symbols:
NCBI Protein Information:breast cancer type 1 susceptibility protein homolog
UniProt Protein Name:Breast cancer type 1 susceptibility protein homolog
UniProt Synonym Protein Names:RING-type E3 ubiquitin transferase BRCA1Curated
Protein Family:Breast cancer type 1 susceptibility protein
UniProt Gene Name:Brca1  
UniProt Entry Name:
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.
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