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Human TATA-box-binding protein (TBP) ELISA Kit (HUEB0613)

SKU:
HUEB0613
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P20226
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
TAF, TATA Box Binding Protein, TBP-Associated Factors
Reactivity:
Human
€649
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Description

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Product Name:Human TATA-box-binding protein (TBP) ELISA Kit
SKU:HUEB0613
Size:96T
Target:Human TATA-box-binding protein (TBP)
Synonyms:TATA sequence-binding protein, TATA-binding factor, TATA-box factor, Transcription initiation factor TFIID TBP subunit, GTF2D1, TF2D, TFIID
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Human
Detection Range:0.312-20ng/mL
Sensitivity:0.169ng/mL
Intra CV:4.9%
Inter CV:7.5%
Linearity:
Sample1:21:41:81:16
Serum(N=5)98-108%82-94%88-98%99-108%
EDTA Plasma(N=5)98-108%87-95%90-100%104-114%
Heparin Plasma(N=5)92-101%105-114%105-115%110-120%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum9892-104
Plasma10094-106
Function:General transcription factor that functions at the core of the DNA-binding multiprotein factor TFIID (PubMed:2374612, PubMed:2363050, PubMed:2194289, PubMed:9836642, PubMed:27193682). Binding of TFIID to the TATA box is the initial transcriptional step of the pre-initiation complex (PIC), playing a role in the activation of eukaryotic genes transcribed by RNA polymerase II (PubMed:2374612, PubMed:2363050, PubMed:2194289, PubMed:9836642, PubMed:27193682). Component of a BRF2-containing transcription factor complex that regulates transcription mediated by RNA polymerase III (PubMed:26638071). Component of the transcription factor SL1/TIF-IB complex, which is involved in the assembly of the PIC (pre-initiation complex) during RNA polymerase I-dependent transcription (PubMed:15970593). The rate of PIC formation probably is primarily dependent on the rate of association of SL1 with the rDNA promoter. SL1 is involved in stabilization of nucleolar transcription factor 1/UBTF on rDNA.
Uniprot:P20226
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant human TATA-box-binding protein
Sub Unit:Binds DNA as monomer (PubMed:2374612, PubMed:2194289). Belongs to the TFIID complex together with the TBP-associated factors (TAFs) (PubMed:9836642, PubMed:27007846). Part of a TFIID-containing RNA polymerase II pre-initiation complex that is composed of TBP and at least GTF2A1, GTF2A2, GTF2E1, GTF2E2, GTF2F1, GTF2H2, GTF2H3, GTF2H4, GTF2H5, GTF2B, TCEA1, ERCC2, ERCC3, TAF1, TAF2, TAF3, TAF4, TAF5, TAF6, TAF7, TAF8, TAF9, TAF10, TAF11, TAF12 and TAF13 (PubMed:27007846). Component of the transcription factor SL1/TIF-IB complex, composed of TBP and at least TAF1A, TAF1B, TAF1C and TAF1D (PubMed:7801123). Association of TBP to form either TFIID or SL1/TIF-IB appears to be mutually exclusive (PubMed:7801123). Interacts with TAF1A, TAF1B and TAF1C (PubMed:7801123). Interacts with TFIIB, NCOA6, DRAP1, DR1 and ELF3 (PubMed:10567404, PubMed:10391676, PubMed:11461703). Interacts with SPIB, SNAPC1, SNAPC2 and SNAPC4 (PubMed:10196196, PubMed:12621023). Interacts with UTF1 (PubMed:9748258). Interacts with BRF2; this interaction promotes recruitment of BRF2 to TATA box-containing promoters (PubMed:11564744, PubMed:26638071). Interacts with UBTF (PubMed:7982918). Interacts with GPBP1 (By similarity). Interacts with CITED2 (By similarity). Interacts with ATF7IP (Probable). Interacts with HIV-1 Tat (PubMed:8121496, PubMed:7608968, PubMed:15719058). Interacts with herpes simplex virus 1 ICP4 (PubMed:8392607). Interacts with human adenovirus E1A protein; this interaction probably disrupts the TBP-TATA complex (PubMed:8146144). Interacts with LLPH (By similarity). Interacts with HSF1 (via transactivation domain) (PubMed:11005381).
Research Area:Neurosciences
Subcellular Location:Nucleus
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:General transcription factor that functions at the core of the DNA-binding multiprotein factor TFIID (PubMed:2374612, PubMed:2363050, PubMed:2194289, PubMed:9836642, PubMed:27193682). Binding of TFIID to the TATA box is the initial transcriptional step of the pre-initiation complex (PIC), playing a role in the activation of eukaryotic genes transcribed by RNA polymerase II (PubMed:2374612, PubMed:2363050, PubMed:2194289, PubMed:9836642, PubMed:27193682). Component of a BRF2-containing transcription factor complex that regulates transcription mediated by RNA polymerase III (PubMed:26638071). Component of the transcription factor SL1/TIF-IB complex, which is involved in the assembly of the PIC (pre-initiation complex) during RNA polymerase I-dependent transcription (PubMed:15970593). The rate of PIC formation probably is primarily dependent on the rate of association of SL1 with the rDNA promoter. SL1 is involved in stabilization of nucleolar transcription factor 1/UBTF on rDNA.
NCBI Summary:Initiation of transcription by RNA polymerase II requires the activities of more than 70 polypeptides. The protein that coordinates these activities is transcription factor IID (TFIID), which binds to the core promoter to position the polymerase properly, serves as the scaffold for assembly of the remainder of the transcription complex, and acts as a channel for regulatory signals. TFIID is composed of the TATA-binding protein (TBP) and a group of evolutionarily conserved proteins known as TBP-associated factors or TAFs. TAFs may participate in basal transcription, serve as coactivators, function in promoter recognition or modify general transcription factors (GTFs) to facilitate complex assembly and transcription initiation. This gene encodes TBP, the TATA-binding protein. A distinctive feature of TBP is a long string of glutamines in the N-terminus. This region of the protein modulates the DNA binding activity of the C terminus, and modulation of DNA binding affects the rate of transcription complex formation and initiation of transcription. The number of CAG repeats encoding the polyglutamine tract is usually 25-42, and expansion of the number of repeats to 45-66 increases the length of the polyglutamine string and is associated with spinocerebellar ataxia 17, a neurodegenerative disorder classified as a polyglutamine disease. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2016]
UniProt Code:P20226
NCBI GenInfo Identifier:1351223
NCBI Gene ID:6908
NCBI Accession:P20226.2
UniProt Secondary Accession:P20226,Q16845, Q6IBM6, Q9UC02, B4E3B3, F5H869,
UniProt Related Accession:P20226
Molecular Weight:35,657 Da
NCBI Full Name:TATA-box-binding protein
NCBI Synonym Full Names:TATA-box binding protein
NCBI Official Symbol:TBP
NCBI Official Synonym Symbols:HDL4; GTF2D; SCA17; TFIID; GTF2D1
NCBI Protein Information:TATA-box-binding protein
UniProt Protein Name:TATA-box-binding protein
UniProt Synonym Protein Names:TATA sequence-binding protein; TATA-binding factor; TATA-box factor; Transcription initiation factor TFIID TBP subunit
Protein Family:TBP-like protein
UniProt Gene Name:TBP
UniProt Entry Name:TBP_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.
ELISAAntibodies
Human TBP (TATA Binding Protein) ELISA Kit (HUES02067)Anti-TBP Antibody (CAB16436)
Anti-TBP Antibody (CAB2192)[KO Validated]
Anti-ACTN1 Antibody (MACO0390)
Anti-BAX Antibody (MACO0392)
TBP Antibody (PACO01610)

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