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Rat E3 ubiquitin-protein ligase HACE1 (Hace1) ELISA Kit (RTEB1535)

SKU:
RTEB1535
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
D3ZBM7
ELISA Type:
Sandwich
Reactivity:
Rat
€649
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Description

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Rat E3 ubiquitin-protein ligase HACE1 (Hace1) ELISA Kit

The Rat HACE1 (E3 Ubiquitin Protein Ligase HACE1) ELISA Kit is a reliable tool for the quantification of HACE1 levels in rat samples, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for various research applications.HACE1 is a critical enzyme involved in the ubiquitination process, playing a crucial role in protein degradation and cellular homeostasis. Dysregulation of HACE1 has been linked to various diseases, including cancer and neurodegenerative disorders, making it a valuable biomarker for understanding disease mechanisms and developing potential therapeutic interventions.

With its user-friendly protocol and superior performance, the Rat HACE1 ELISA Kit is an essential tool for researchers studying the role of HACE1 in health and disease, providing valuable insights into its functions and potential implications for future research and clinical applications.

Product Name:Rat E3 ubiquitin-protein ligase HACE1 (Hace1) ELISA Kit
SKU:RTEB1535
Size:96T
Target:Rat E3 ubiquitin-protein ligase HACE1 (Hace1)
Synonyms:HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1, HECT-type E3 ubiquitin transferase HACE1
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Rat
Detection Range:0.312-20ng/mL
Sensitivity:0.172ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:E3 ubiquitin-protein ligase involved in Golgi membrane fusion and regulation of small GTPases. Acts as a regulator of Golgi membrane dynamics during the cell cycle: recruited to Golgi membrane by Rab proteins and regulates postmitotic Golgi membrane fusion. Acts by mediating ubiquitination during mitotic Golgi disassembly, ubiquitination serving as a signal for Golgi reassembly later, after cell division. Specifically interacts with GTP-bound RAC1, mediating ubiquitination and subsequent degradation of active RAC1, thereby playing a role in host defense against pathogens. May also act as a transcription regulator via its interaction with RARB.
Uniprot:D3ZBM7
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant rat E3 ubiquitin-protein ligase HACE1
Sub Unit:Interacts with RARB. Interacts with RAB1 (RAB1A, RAB1B or RAB1C), RAB4 (RAB4A or RAB4B) and RAB11 (RAB11A or RAB11B); in a GTP-dependent manner. Interacts with RAC1; in a GTP-dependent manner. Interacts with the 26S proteasomal complex through the 20S core proteasomal subunit.
Research Area:Cancer
Subcellular Location:Golgi apparatus Golgi stack membrane Cytoplasm Endoplasmic reticulum A significant portion localizes to the endoplasmic reticulum. Targeted to Golgi membrane via its interaction with Rab proteins.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:E3 ubiquitin-protein ligase involved in Golgi membrane fusion and regulation of small GTPases. Acts as a regulator of Golgi membrane dynamics during the cell cycle: recruited to Golgi membrane by Rab proteins and regulates postmitotic Golgi membrane fusion. Acts by mediating ubiquitination during mitotic Golgi disassembly, ubiquitination serving as a signal for Golgi reassembly later, after cell division. Specifically interacts with GTP-bound RAC1, mediating ubiquitination and subsequent degradation of active RAC1, thereby playing a role in host defense against pathogens. May also act as a transcription regulator via its interaction with RARB.
UniProt Protein Details:
NCBI Summary:
UniProt Code:D3ZBM7
NCBI GenInfo Identifier:158711729
NCBI Gene ID:361866
NCBI Accession:NP_001102009.2
UniProt Secondary Accession:D3ZBM7,D3ZBM8
UniProt Related Accession:D3ZBM7
Molecular Weight:102,159 Da
NCBI Full Name:E3 ubiquitin-protein ligase HACE1
NCBI Synonym Full Names:HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1
NCBI Official Symbol:Hace1
NCBI Official Synonym Symbols:
NCBI Protein Information:E3 ubiquitin-protein ligase HACE1
UniProt Protein Name:E3 ubiquitin-protein ligase HACE1
UniProt Synonym Protein Names:HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1; HECT-type E3 ubiquitin transferase HACE1
Protein Family:E3 ubiquitin-protein ligase
UniProt Gene Name:Hace1
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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