Human R-Spondin1 Recombinant

$160.00 – $2,750.00

SKU: RKQ2MKA7 Tags: hRspo1, Q2MKA7, R-SPONDIN-1, RSPO1

Description

Accession

Q2MKA7

Source

Optimized DNA sequence encoding peptidase domain of Human R-Spondin1(SER21-ALA263 ) including a C-terminal His tag was expressed in HEK293 cells.

Molecular weight

Recombinant human R-Spondin-1 is a protein consisting of 433 amino acid residue subunits,due to glycosylation migrates as an approximately 53kDa protein on reduced SDS-PAGE.

Purity

>95%, as determined by SDS-PAGE and HPLC

Biological Activity

The activity was tested by the ability of immobilized recombinant human RSPO1 to bind recombinant human LIMP2 with a linear range of 30-500 ng/ml

Endotoxin

Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than 0.1 ng/µg(1EU/µg).

Presentation

Recombinant Human R-spondin1 is lyophilized from 0.2 μm filtered PBS solution, pH7.2 , 5% Trehalose.

Storage

RecombinantR-spondin1can be stored in working aliquots at 2° - 8° C for one month, or at -20°C to-70°Cfor twelve months. Avoid repeated freeze/thaw cycles.

Usage

This product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Interactor

Q9ULT6

Biological Process

Sensory-transduction

Molecular function

Heparin-binding

Methods

Crypt isolation, cell lines and culture conditions

Isolation and culture of intestinal crypts were done as described previously with modifications indicated.
Briefly, the small intestine of 4- to 12-week-old C57BL/6 wild-type or Bim-deficient mice was opened longitudinally and villi were scraped off using a coverslip.
The intestine was cut into 1–2 cm pieces, washed three times with cold PBS and incubated with 2 mM EDTA in PBS for 30 min at 4 °C on a rotating wheel.
Residual villi were removed by gentle shaking, the villi containing supernatant was removed and replaced with cold PBS.
This procedure was repeated until no villi could be observed anymore in the supernatant using microscopy.
Crypts were then detached from the basal membrane by vigorous shaking.
The crypts enriched in the supernatant were passed through a 70-μm cell strainer , centrifuged at 100 × g (3 min, 4 °C) and resuspended in 10 ml PBS for counting using microscopy.
Pelleted crypts were resuspended in Matrigel at a desired crypt density.
Two hundred to five hundred…

Isolation and culture of intestinal crypts were done as described previously with modifications indicated.
Briefly, the small intestine of 4- to 12-week-old C57BL/6 wild-type or Bim-deficient mice was opened longitudinally and villi were scraped off using a coverslip.
The intestine was cut into 1–2 cm pieces, washed three times with cold PBS and incubated with 2 mM EDTA in PBS for 30 min at 4 °C on a rotating wheel.
Residual villi were removed by gentle shaking, the villi containing supernatant was removed and replaced with cold PBS.
This procedure was repeated until no villi could be observed anymore in the supernatant using microscopy.
Crypts were then detached from the basal membrane by vigorous shaking.
The crypts enriched in the supernatant were passed through a 70-μm cell strainer , centrifuged at 100 × g (3 min, 4 °C) and resuspended in 10 ml PBS for counting using microscopy.
Pelleted crypts were resuspended in Matrigel at a desired crypt density.
Two hundred to five hundred crypts in 7 μl Matrigel were seeded per well on a pre-warmed 96-well flat-bottom plate and incubated for 15 min at 37 °C.
Then, 70 μl of complete crypt culture medium was added medium'>(ADF medium: advanced DMEM/F12 , 0.1% BSA , 2 mM L-glutamine , 10 mM HEPES , 100 U/ml penicillin , 100 μg/ml streptomycin , 20 μg/ml nystatin , 1 mM N-acetyl cysteine , 1 × B27 supplement , 1 × N2 supplement , 50 ng/ml mEGF , 100 ng/ml mNoggin , 500 ng/ml human R-spondin-1 .
Alternatively, hR-spondin-1 was added as conditioned medium'>medium of medium'>hR-spondin-1-transfected medium'>HEK 293T cells to a final volume of 10% crypt culture medium'>medium.
In some experiments, hR-spondin-1 was omitted.
Organoids were cultured at 37 °C in a 5% CO₂ atmosphere for at least 3 days before stimulation of cell death.
The human colorectal tumor cell line Caco-2 and the murine intestinal tumor cell line MC38 were obtained from , and cultured in IMDM supplemented with 10% FCS (PAA), 1 × MEM amino-acid solution , 4 mM L-glutamine and 50 μg/ml gentamycin at 37 °C and 5% CO₂.

Human R-Spondin1 Recombinant

Description

Methods

Crypt isolation, cell lines and culture conditions

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