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Rat B-cell linker protein (Blnk) ELISA Kit (RTEB1119)

SKU:
RTEB1119
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q4KM52
ELISA Type:
Sandwich
Reactivity:
Rat
€649
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Description

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Rat B-cell linker protein (Blnk) ELISA Kit

The Rat B-Cell Linker Protein (BLNK) ELISA Kit is specially designed for the precise and accurate detection of BLNK levels in rat samples, including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring dependable and consistent results for a variety of research purposes.BLNK is a key protein involved in B cell receptor signaling, serving as a crucial linker between the receptor and downstream signaling pathways.

Its role in regulating B cell activation and differentiation makes it a valuable marker for studying immune responses, autoimmune diseases, and lymphoid malignancies.With its reliable performance and broad research applications, the Rat BLNK ELISA Kit is an essential tool for scientists investigating B cell biology and immunological processes in rat models. Unlock new insights into immune function and disease mechanisms with this innovative ELISA kit from Assay Genie.

Product Name:Rat B-cell linker protein (Blnk) ELISA Kit
SKU:RTEB1119
Size:96T
Target:Rat B-cell linker protein (Blnk)
Synonyms:Cytoplasmic adapter protein
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Rat
Detection Range:0.156-10ng/mL
Sensitivity:0.081ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Functions as a central linker protein, downstream of the B-cell receptor (BCR), bridging the SYK kinase to a multitude of signaling pathways and regulating biological outcomes of B-cell function and development. Plays a role in the activation of ERK/EPHB2, MAP kinase p38 and JNK. Modulates AP1 activation. Important for the activation of NF-kappa-B and NFAT. Plays an important role in BCR-mediated PLCG1 and PLCG2 activation and Ca(2+) mobilization and is required for trafficking of the BCR to late endosomes. However, does not seem to be required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signaling. May be required for the RAC1-JNK pathway. Plays a critical role in orchestrating the pro-B cell to pre-B cell transition. May play an important role in BCR-induced B-cell apoptosis.
Uniprot:Q4KM52
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant rat B-cell linker protein
Sub Unit:Associates with PLCG1, VAV1 and NCK1 in a B-cell antigen receptor-dependent fashion. Interacts with VAV3, PLCG2 and GRB2. Interacts through its SH2 domain with CD79A. Interacts (via SH2 domain) with SYK; phosphorylated and activated by SYK. Interacts with SCIMP.
Subcellular Location:Cytoplasm Cell membrane BCR activation results in the translocation to membrane fraction.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:BLNK: an adaptor protein that bridges the B-cell receptor-associated kinases (BCR) with a multitude of signaling pathways, regulating biologic outcomes of B-cell function and development. Plays an important role in BCR-mediated PLCG1 activation and Ca(2 ) mobilization. Does not seem to be not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signaling. Plays a critical role in orchestrating the pro-B cell to pre-B cell transition. Following BCR activation, phosphorylated on tyrosine residues by SYK and LYN. When phosphorylated, serves as a scaffold to assemble downstream targets of antigen activation, including PLCG1, VAV1, GRB2 and NCK1. Its phosphorylation is required for both Ca(2 ) and MAPK signaling pathways. Defects in BLNK are the cause of hypoglobulinemia and absent B-cells.It has tumor supressor activity that is lost in many cases of childhood pre-B acute lymphoblastic leukemia (ALL). Two alternatively spliced isoforms have been described; these are differentially involved in activation and apoptosis of B lymphocytes.Protein type: Adaptor/scaffoldCellular Component: cytoplasm; plasma membraneMolecular Function: SH3/SH2 adaptor activity; transmembrane receptor protein tyrosine kinase adaptor protein activityBiological Process: B cell activation; immune response; positive regulation of signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway
UniProt Protein Details:
NCBI Summary:
UniProt Code:Q4KM52
NCBI GenInfo Identifier:71043856
NCBI Gene ID:499356
NCBI Accession:NP_001020938.1
UniProt Secondary Accession:Q4KM52
UniProt Related Accession:Q4KM52
Molecular Weight:50,634 Da
NCBI Full Name:B-cell linker protein
NCBI Synonym Full Names:B-cell linker
NCBI Official Symbol:Blnk
NCBI Official Synonym Symbols:
NCBI Protein Information:B-cell linker protein
UniProt Protein Name:B-cell linker protein
UniProt Synonym Protein Names:Cytoplasmic adapter protein
Protein Family:B-cell linker protein
UniProt Gene Name:Blnk
UniProt Entry Name:BLNK_RAT
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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