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Human AP-2 complex subunit beta (AP2B1) ELISA Kit (HUEB1289)

SKU:
HUEB1289
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P63010
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
AP2B1, AP-2 complex subunit beta, Clathrin assembly protein complex 2 beta large chain
Reactivity:
Human
€649
Frequently bought together:

Description

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Human AP-2 complex subunit beta (AP2B1) ELISA Kit

The Human AP-2 Complex Subunit Beta (AP2B1) ELISA Kit is specifically designed for the accurate quantification of AP2B1 levels in human samples, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a wide range of research applications.AP2B1 is a key component of the AP-2 complex, which plays a critical role in clathrin-mediated endocytosis and intracellular trafficking. Dysregulation of AP2B1 has been linked to various diseases, including cancer, neurodegenerative disorders, and infectious diseases.

Therefore, the detection of AP2B1 levels is important for understanding the underlying mechanisms of these conditions and developing potential therapeutic interventions.Overall, the Human AP-2 Complex Subunit Beta (AP2B1) ELISA Kit is a valuable tool for researchers studying endocytic pathways, intracellular trafficking, and disease mechanisms associated with AP2B1 dysregulation. Its high sensitivity and accuracy make it a reliable choice for precise quantification of AP2B1 levels in human samples.

Product Name:Human AP-2 complex subunit beta (AP2B1) ELISA Kit
SKU:HUEB1289
Size:96T
Target:Human AP-2 complex subunit beta (AP2B1)
Synonyms:AP105B, Adaptor protein complex AP-2 subunit beta, Adaptor-related protein complex 2 subunit beta, Beta-2-adaptin, Beta-adaptin, Clathrin assembly protein complex 2 beta large chain, Plasma membrane adaptor HA2/AP2 adaptin beta subunit, ADTB2, CLAPB1
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Human
Detection Range:0.312-20ng/mL
Sensitivity:0.11ng/ml
Intra CV:5.6%
Inter CV:9.2%
Linearity:
Sample1:21:41:81:16
Serum(N=5)110-120%96-107%87-96%110-119%
EDTA Plasma(N=5)84-84%113-122%102-111%107-117%
Heparin Plasma(N=5)108-118%114-123%105-116%100-100%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum9791-103
Plasma9993-105
Function:Component of the adaptor protein complex 2 (AP-2). Adaptor protein complexes function in protein transport via transport vesicles in different membrane traffic pathways. Adaptor protein complexes are vesicle coat components and appear to be involved in cargo selection and vesicle formation. AP-2 is involved in clathrin-dependent endocytosis in which cargo proteins are incorporated into vesicles surrounded by clathrin (clathrin-coated vesicles, CCVs) which are destined for fusion with the early endosome. The clathrin lattice serves as a mechanical scaffold but is itself unable to bind directly to membrane components. Clathrin-associated adaptor protein (AP) complexes which can bind directly to both the clathrin lattice and to the lipid and protein components of membranes are considered to be the major clathrin adaptors contributing the CCV formation. AP-2 also serves as a cargo receptor to selectively sort the membrane proteins involved in receptor-mediated endocytosis. AP-2 seems to play a role in the recycling of synaptic vesicle membranes from the presynaptic surface. AP-2 recognizes Y-X-X-[FILMV] (Y-X-X-Phi) and [ED]-X-X-X-L-[LI] endocytosis signal motifs within the cytosolic tails of transmembrane cargo molecules. AP-2 may also play a role in maintaining normal post-endocytic trafficking through the ARF6-regulated, non-clathrin pathway. The AP-2 beta subunit acts via its C-terminal appendage domain as a scaffolding platform for endocytic accessory proteins; at least some clathrin-associated sorting proteins (CLASPs) are recognized by their [DE]-X(1,2)-F-X-X-[FL]-X-X-X-R motif. The AP-2 beta subunit binds to clathrin heavy chain, promoting clathrin lattice assembly; clathrin displaces at least some CLASPs from AP2B1 which probably then can be positioned for further coat assembly.
Uniprot:P63010
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant human AP-2 complex subunit beta
Sub Unit:Adaptor protein complex 2 (AP-2) is a heterotetramer composed of two large adaptins (alpha-type subunit AP2A1 or AP2A2 and beta-type subunit AP2B1), a medium adaptin (mu-type subunit AP2M1) and a small adaptin (sigma-type subunit AP2S1). Interacts with EPN1. Interacts with EPS15; clathrin competes with EPS15. Interacts with SNAP91; clathrin competes with SNAP91. Interacts with CLTC; clathrin competes with EPS15, SNAP91 and PIP5K1C. Interacts with LDLRAP1. Interacts with AMPH and BIN1. Interacts with ARF6 (GDP-bound). Interacts (dephosphorylated at Tyr-737) with ARRB1; phosphorylation of AP2B1 at Tyr-737 disrupts the interaction. Interacts with SLC2A8. Interacts with SCYL1 and SCYL2. Interacts with TGFBR1 and TGFBR2. Interacts with PIP5K1C; clathrin competes with PIP5K1C (By similarity). Interacts with DENND1B, but not with DENND1A, nor DENND1C. Interacts with FCHO1.
Research Area:Neurosciences
Subcellular Location:Cell membrane Membrane Coated pit Peripheral membrane protein Cytoplasmic side AP-2 appears to be excluded from internalizing CCVs and to disengage from sites of endocytosis seconds before internalization of the nascent CCV.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:AP2B1: Component of the adaptor protein complex 2 (AP-2). Adaptor protein complexes function in protein transport via transport vesicles in different membrane traffic pathways. Adaptor protein complexes are vesicle coat components and appear to be involved in cargo selection and vesicle formation. AP-2 is involved in clathrin-dependent endocytosis in which cargo proteins are incorporated into vesicles surrounded by clathrin (clathrin- coated vesicles, CCVs) which are destined for fusion with the early endosome. The clathrin lattice serves as a mechanical scaffold but is itself unable to bind directly to membrane components. Clathrin-associated adaptor protein (AP) complexes which can bind directly to both the clathrin lattice and to the lipid and protein components of membranes are considered to be the major clathrin adaptors contributing the CCV formation. AP-2 also serves as a cargo receptor to selectively sort the membrane proteins involved in receptor-mediated endocytosis. AP-2 seems to play a role in the recycling of synaptic vesicle membranes from the presynaptic surface. AP-2 recognizes Y-X-X-[FILMV] (Y-X-X-Phi) and [ED]-X-X-X-L-[LI] endocytosis signal motifs within the cytosolic tails of transmembrane cargo molecules. AP-2 may also play a role in maintaining normal post-endocytic trafficking through the ARF6-regulated, non-clathrin pathway. The AP-2 beta subunit acts via its C-terminal appendage domain as a scaffolding platform for endocytic accessory proteins; at least some clathrin- associated sorting proteins (CLASPs) are recognized by their [DE]- X(1,2)-F-X-X-[FL]-X-X-X-R motif. The AP-2 beta subunit binds to clathrin heavy chain, promoting clathrin lattice assembly; clathrin displaces at least some CLASPs from AP2B1 which probably then can be positioned for further coat assembly. Belongs to the adaptor complexes large subunit family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Vesicle

Chromosomal Location of Human Ortholog: 17q12

Cellular Component: intracellular membrane-bound organelle; membrane; plasma membrane; AP-2 adaptor complex; cytosol

Molecular Function:clathrin binding; protein binding; signal sequence binding; protein transporter activity

Biological Process: epidermal growth factor receptor signaling pathway; negative regulation of epidermal growth factor receptor signaling pathway; synaptic transmission; intracellular protein transport; axon guidance; nerve growth factor receptor signaling pathway; viral reproduction; ephrin receptor signaling pathway; antigen processing and presentation of exogenous peptide antigen via MHC class II; regulation of defense response to virus by virus

NCBI Summary:The protein encoded by this gene is one of two large chain components of the assembly protein complex 2, which serves to link clathrin to receptors in coated vesicles. The encoded protein is found on the cytoplasmic face of coated vesicles in the plasma membrane. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:P63010
NCBI GenInfo Identifier:51702211
NCBI Gene ID:163
NCBI Accession:P63010.1
UniProt Secondary Accession:P63010,P21851, Q7Z451, Q96J19, A6NJP3,
UniProt Related Accession:P63010
Molecular Weight:937
NCBI Full Name:AP-2 complex subunit beta
NCBI Synonym Full Names:adaptor-related protein complex 2, beta 1 subunit
NCBI Official Symbol:AP2B1
NCBI Official Synonym Symbols:ADTB2; AP105B; CLAPB1; AP2-BETA
NCBI Protein Information:AP-2 complex subunit beta; beta-adaptin; beta-2-adaptin; adaptin, beta 2 (beta); adaptor protein complex AP-2 subunit beta; adapter-related protein complex 2 beta subunit; adapter-related protein complex 2 subunit beta; adaptor-related protein complex 2 s
UniProt Protein Name:AP-2 complex subunit beta
UniProt Synonym Protein Names:AP105B; Adaptor protein complex AP-2 subunit beta; Adaptor-related protein complex 2 subunit beta; Beta-2-adaptin; Beta-adaptin; Clathrin assembly protein complex 2 beta large chain; Plasma membrane adaptor HA2/AP2 adaptin beta subunit
Protein Family:AP-2 complex
UniProt Gene Name:AP2B1
UniProt Entry Name:AP2B1_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.
ELISA
Human AP2B1 / AP-2 complex subunit beta ELISA Kit

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