The Mouse SUMO Conjugating Enzyme UBC9 (UBE2I) ELISA Kit is a powerful tool for quantifying levels of SUMO conjugating enzyme UBC9 in mouse serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and reproducible results for a wide range of research studies.SUMO conjugating enzyme UBC9 is a key player in the SUMOylation process, regulating protein function, localization, and stability. Dysregulation of SUMOylation has been implicated in various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions.
Therefore, measuring UBC9 levels can provide valuable insights into disease mechanisms and potential therapeutic targets.With its user-friendly assay procedure and reliable performance, the Mouse SUMO Conjugating Enzyme UBC9 ELISA Kit is an indispensable tool for researchers investigating the role of SUMOylation in health and disease.
RING-type E3 SUMO transferase UBC9, SUMO-protein ligase, Ubiquitin carrier protein 9, Ubiquitin carrier protein I, Ubiquitin-conjugating enzyme E2 I, Ubiquitin-protein ligase I, mUBC9, Ubc9, Ubce2i, Ubce9
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.312-20ng/mL
Sensitivity:
0.159ng/mL
Intra CV:
5.1%
Inter CV:
7.5%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
100-113%
91-100%
103-113%
97-107%
EDTA Plasma(N=5)
98-106%
108-117%
82-92%
110-120%
Heparin Plasma(N=5)
85-94%
107-115%
83-96%
87-99%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
108
102-114
Plasma
110
104-116
Function:
Accepts the ubiquitin-like proteins SUMO1, SUMO2 and SUMO3 from the UBLE1A-UBLE1B E1 complex and catalyzes their covalent attachment to other proteins with the help of an E3 ligase such as RANBP2, CBX4 and ZNF451. Can catalyze the formation of poly-SUMO chains. Essential for nuclear architecture, chromosome segregation and embryonic viability. Necessary for sumoylation of FOXL2 and KAT5 (By similarity). Sumoylates p53/TP53 at 'Lys-386'.
Uniprot:
P63280
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse SUMO-conjugating enzyme UBC9
Sub Unit:
Forms a tight complex with RANGAP1 and RANBP2. Interacts with SIAH1 and PARP. Interacts with various transcription factors such as TFAP2A, TFAP2B, TFAP2C, AR, ETS1 and SOX4. Interacts with RASD2. Interacts with RWDD3; the interaction enhances the sumoylation of a number of proteins such as HIF1A and I-kappa-B. Interacts with FOXL2. Forms a complex with SENP6 and UBE2I in response to UV irradiation. Interacts with DNM1L (via its GTPase and B domains); the interaction promotes sumoylation of DNM1L, mainly in the B domain (By similarity). Interacts with HIPK1, HIPK2, PPM1J and TCF3. Interacts with Moloney murine leukemia virus CA and adenovirus type 5 and type 12 E1A. Interacts with NR2C1; the interaction promotes its sumoylation. Interacts with NFATC2IP; this inhibits formation of poly-SUMO chains. Interacts with FHIT (By similarity). Interacts with PRKRA and p53/TP53. Interacts with UHRF2. Interacts with NR3C1 and this interaction is enhanced in the presence of RWDD3. Interacts with MTA1 (By similarity). Interacts with ZNF451. Identified in a complex with SUMO2 and UBC9, where one ZNF451 interacts with one UBC9 and two SUMO2 chains, one bound to the UBC9 active site and the other to another region of the same UBC9 molecule. Interacts with SETX (By similarity). Interacts with CPEB3 (PubMed:26074071).
Research Area:
Epigenetics
Subcellular Location:
Nucleus Cytoplasm Mainly nuclear. In spermatocytes, localizes in synaptonemal complexes. Recruited by BCL11A into the nuclear body.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
UniProt Protein Details:
NCBI Summary:
The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. This gene encodes a member of the E2 ubiquitin-conjugating enzyme family. Four alternatively spliced transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Jul 2008]
SUMO-protein ligase; Ubiquitin carrier protein 9; Ubiquitin carrier protein I; Ubiquitin-conjugating enzyme E2 I; Ubiquitin-protein ligase I; p18
Protein Family:
UniProt Gene Name:
UBE2I
UniProt Entry Name:
UBC9_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.
