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SF1 (Phospho-Ser82) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01318
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
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Description

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SF1 (Phospho-Ser82)Colorimetric Cell-Based ELISA Kit

The SF1 (Phospho-Ser82) Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the precise measurement of SF1 phosphorylation at serine 82 in cell lysates. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for your research needs.SF1, also known as splicing factor 1, plays a crucial role in alternative splicing, gene regulation, and RNA processing. Phosphorylation of SF1 at serine 82 has been linked to various cellular processes and diseases, making it a valuable target for studying signal transduction pathways and potential therapeutic interventions.

With the SF1 (Phospho-Ser82) Colorimetric Cell-Based ELISA Kit, researchers can explore the role of SF1 phosphorylation in cellular functions and disease pathways, paving the way for new insights and discoveries in the field of molecular biology and drug development.

Product Name:SF1 (Phospho-Ser82) Colorimetric Cell-Based ELISA
Product Code:CBCAB01318
ELISA Type:Cell-Based
Target:SF1 (Phospho-Ser82)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The SF1 (Phospho-Ser82) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SF1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SF1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SF1 phosphorylation.

Qualitative determination of SF1 (Phospho-Ser82) concentration is achieved by an indirect ELISA format. In essence, SF1 (Phospho-Ser82) is captured by SF1 (Phospho-Ser82)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 7536, UniProt ID: Q15637, OMIM: 601516, Unigene: Hs.502829/Hs.708209
Gene Symbol:SF1
Sub Type:Phospho
UniProt Protein Function:SF1: a splicing factor necessary for the ATP-dependent first step of spliceosome assembly. Binds to the intron branch point sequence (BPS) 5'-UACUAAC-3' of the pre-mRNA. May act as transcription repressor. Phosphorylation of SF1 by cGMP-dependent protein kinase regulates spliceosome assembly. Binds U2AF2. Interacts with U1 snRNA. Binds EWSR1, FUS and TAF15. Six alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Transcription, coactivator/corepressor; Spliceosome; RNA splicing; RNA-binding

Chromosomal Location of Human Ortholog: 11q13

Cellular Component: nucleoplasm; spliceosome; ribosome; nucleus

Molecular Function:zinc ion binding; RNA binding; transcription corepressor activity

Biological Process: nuclear mRNA 3'-splice site recognition; nuclear mRNA splicing, via spliceosome; Leydig cell differentiation; regulation of transcription, DNA-dependent; transcription, DNA-dependent; negative regulation of smooth muscle cell proliferation; male sex determination; spliceosome assembly; regulation of steroid biosynthetic process

NCBI Summary:This gene encodes a nuclear pre-mRNA splicing factor. The encoded protein specifically recognizes the intron branch point sequence and is required for the early stages of spliceosome assembly. Alternate splicing results in multiple transcript variants. [provided by RefSeq, May 2010]
UniProt Code:Q15637
NCBI GenInfo Identifier:38258418
NCBI Gene ID:7536
NCBI Accession:Q15637.4
UniProt Secondary Accession:Q15637,Q14818, Q14819, Q15913, Q8IY00, Q92744, Q92745 Q969H7, Q9BW01, Q9UEI0, B7Z1Q1, C9JJE2,
UniProt Related Accession:Q15637
Molecular Weight:65,371 Da
NCBI Full Name:Splicing factor 1
NCBI Synonym Full Names:splicing factor 1
NCBI Official Symbol:SF1  
NCBI Official Synonym Symbols:BBP; MBBP; ZFM1; ZNF162; D11S636; ZCCHC25  
NCBI Protein Information:splicing factor 1; zinc finger protein 162; transcription factor ZFM1; zinc finger gene in MEN1 locus; mammalian branch point-binding protein
UniProt Protein Name:Splicing factor 1
UniProt Synonym Protein Names:Mammalian branch point-binding protein; BBP; mBBP; Transcription factor ZFM1; Zinc finger gene in MEN1 locus; Zinc finger protein 162
Protein Family:Splicing factor
UniProt Gene Name:SF1  
UniProt Entry Name:SF01_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.Incubate the cells for overnight at 37 °C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-SF1 (Phospho-Ser82) Antibody, Anti-SF1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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