Mouse Noggin Recombinant

Mouse Noggin Recombinant

$70.00$3,500.00

SKU: RKP97466 Category: Tags: ,

accession P97466


Source Optimized DNA sequence encoding Mouse Noggin mature chain was expressed in Escherichia Coli.
Molecular weight Native Mouse Noggin, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 23 kDa. Recombinant Noggin is a disulfide-linked homodimer protein consisting of 2x206 amino acid residue subunits, migrates as an approximately 46kDa protein under non-reducing and as 23kDa under reducing conditions in SDS-PAGE.
Purity >96%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by induced alkaline phosphatase secretion in ATDC cellsand was determined to be ≤ ng/ml, corresponding to a specific activity of ≥x units/mg.
Protein Sequence MERCPSLGVT LYALVVVLGL RAAPAGGQHY LHIRPAPSDN LPLVDLIEHP DPIFDPKEKD LNETLLRSLL GGHYDPGFMA TSPPEDRPGG GGGPAGGAED LAELDQLLRQ RPSGAMPSEI KGLEFSEGLA QGKKQRLSKK LRRKLQMWLW SQTFCPVLYA WNDLGSRFWP RYVKVGSCFS KRSCSVPEGM VCKPSKSVHL TVLRWRCQRR GGQRCGWIPI QYPIISECKC SC
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation Mouse Noggin was lyophilized from a.2 μm filtered solution in PBS, pH.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Biological Process Chondrogenesis
Biological Process Differentiation
Molecular function Developmental-protein

Methods

Isolation of intestinal crypts

  • Tissue was removed from PBS solution and washed multiple times with ice-cold PBS washes until the solution remained clear.
  • The specimen was then placed in a Petri dish containing PBS on ice with the mucosal surface facing upward.
  • Using a razor blade, excess mucoid material was scrapped from the epithelial surface.
  • The specimen was then divided into approximately 0.5 cm2 pieces.
  • These pieces were placed into a 2.5 mmol/L EDTA solution in PBS for 30 minutes of incubation with gentle shaking at 4°C.
  • After this incubation period, the fragments were allowed to settle and the supernatant was discarded.
  • 10 ml of cold PBS was added to the sample, and subsequently vortexed for 10 seconds with 1-second bursts.
  • The fragments were allowed to settle, and the supernatant was removed and saved on ice.
  • Again 10 ml of PBS were added and the process was repeated eight times.
  • Samples were spun down at 100 g for 2 minutes.
  • The supernatant…
  • Tissue was removed from PBS solution and washed multiple times with ice-cold PBS washes until the solution remained clear.
  • The specimen was then placed in a Petri dish containing PBS on ice with the mucosal surface facing upward.
  • Using a razor blade, excess mucoid material was scrapped from the epithelial surface.
  • The specimen was then divided into approximately 0.5 cm2 pieces.
  • These pieces were placed into a 2.5 mmol/L EDTA solution in PBS for 30 minutes of incubation with gentle shaking at 4°C.
  • After this incubation period, the fragments were allowed to settle and the supernatant was discarded.
  • 10 ml of cold PBS was added to the sample, and subsequently vortexed for 10 seconds with 1-second bursts.
  • The fragments were allowed to settle, and the supernatant was removed and saved on ice.
  • Again 10 ml of PBS were added and the process was repeated eight times.
  • Samples were spun down at 100 g for 2 minutes.
  • The supernatant was discarded.
  • The contents of the pellets were examined under light microscopy using a Nikon TMS microscope to assess purity of crypt fractions.
  • Typically, all fractions were pooled together to increase yield of epithelial crypts.
  • The pooled fractions were then purified using a 100-µm pore filter .
  • Fetal Bovine Serum at 10% per volume was then used to suspend the contents of the filtrate.
  • These clusters were examined under light microscopy and counted.
  • 500 crypt clusters were suspended in 50 µL Matrigel as previously described in Sato's 3-D Matrigel culture system developed for murine intestines

Read more

Monolayer neuronal differentiation

  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing…
  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing the same ligands over 7 days (75% medium'>medium'>KSR and 25% medium'>N2B27 first day, 50% of each third day, and 25% medium'>medium'>KSR with 75% medium'>N2B27 fifth day).
  • Once established in N2B27 conditions, Wnt1, Noggin, SB431542 and −1 BDNF , 0.2 mM ascorbic acid and 100 ng ml−1 FGF8 were added.
  • Three days later, cells were dissociated with Hank's buffered saline solution for 1 h at room temperature, and lifted mechanically, then replated en bloc on to poly-l-ornithine/laminin-coated plasticware.
  • Neuronal maturation ensued with BDNF and ascorbic acid as before, supplemented with 10 ng ml−1 GDNF , 1 ng ml−1 TGFβ3 and 0.5 mM dibutyryl-cAMP for the next 7 days.
  • After a total of 23–31 days, the resulting neuronal cultures were analysed by immunocytochemistry, qPCR and western blot.

Read more

Cell culture, transformation assays and luciferase assays

  • All cell lines (NIH3T3, HEK 293, BJ-ET-st p53,p16, SW480 and HCT-116) were cultured in DMEM supplemented with 10% fetal bovine serum and glutamine/penicillin/streptomycin.
  • For focus formation assays, NIH3T3 cells were transfected using lipofectamine with 200 ng of pBabe-RasV12 plasmid DNA together with 200 ng of pRS shRNA plasmid DNA designed against Nrbp1 (the sequence of shRNA hairpins is provided in the 5 per 6-cm plate and fed every 4 days for 2 weeks and then stained with methylene blue and foci were counted.
  • Each experiment involved five replicate transfections performed on three separate occasions.
  • BJ-ET-st p53,p16 soft agar assays were performed as described previously (U-test.
  • Luciferase assays were performed in 24-well plates with 7.5 × 103 HCT-116 or HEK 293 cells.
  • Cells were cotransfected using lipofectamine with either 200 ng NRBP1 shRNA vectors or empty vector in combination with either 150 ng M50 Super 8x TOPFlash or M51 Super 8 × FOPFlash and 50 ng…
  • All cell lines (NIH3T3, HEK 293, BJ-ET-st p53,p16, SW480 and HCT-116) were cultured in DMEM supplemented with 10% fetal bovine serum and glutamine/penicillin/streptomycin.
  • For focus formation assays, NIH3T3 cells were transfected using lipofectamine with 200 ng of pBabe-RasV12 plasmid DNA together with 200 ng of pRS shRNA plasmid DNA designed against Nrbp1 (the sequence of shRNA hairpins is provided in the 5 per 6-cm plate and fed every 4 days for 2 weeks and then stained with methylene blue and foci were counted.
  • Each experiment involved five replicate transfections performed on three separate occasions.
  • BJ-ET-st p53,p16 soft agar assays were performed as described previously (U-test.
  • Luciferase assays were performed in 24-well plates with 7.5 × 103 HCT-116 or HEK 293 cells.
  • Cells were cotransfected using lipofectamine with either 200 ng NRBP1 shRNA vectors or empty vector in combination with either 150 ng M50 Super 8x TOPFlash or M51 Super 8 × FOPFlash and 50 ng Renilla (pRL-SV40) plasmid.
  • At 72  h after transfection, cells were lysed in 100-μl passive lysis buffer, and the dual luciferase assay performed according to the manufacturers protocol .
  • The relative luciferase activity was calculated against Renilla activity.
  • Each shRNA was assayed in experiments involving six replicates and these experiments were performed on at least two separate occasions.
  • Data were statistically analysed using ANOVA.
  • CellSensor assays were performed in 96-well plates with 2.4 × 104 SW480 cells.
  • Cells were transiently transfected with 20 nm siRNA using Lipofectamine RNAimax .
  • At 72 h after transfection FRET read-out was measured using beta-lactamase loading solution by standard procedures.
  • Data were statistically analysed using ANOVA.
  • Ex vivo crypts were isolated from Nrbp cKO and control mice small intestine by incubating in 2 mM EDTA in PBS.
  • Crypt cultures were grown in Matrigel and provided with Advanced MEM/F23 supplemented with N2 , B27 , EGF , NOGGIN and m-Spondin (

Read more

Crypt isolation and organoid culture

  • Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
  • For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
  • In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
  • Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
  • Organoid growth was monitored by light microscopy.
  • In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml),…
  • Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
  • For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
  • In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
  • Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
  • Organoid growth was monitored by light microscopy.
  • In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml), necrostatin-1 (30 μM) or caspase-8 inhibitor (50 μM ).
  • Cell Viability of organoids was analyzed indirectly by quantification of relative ATP level with the CellTiter-Glo assay fromaccording to the manufacturer's instructions.
  • Luminescence was measured on the microplate reader infinite M200 .

Read more

Crypt isolation and organoid culture

  • Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
  • For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
  • In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
  • Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
  • Organoid growth was monitored by light microscopy.
  • In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml),…
  • Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
  • For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
  • In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
  • Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
  • Organoid growth was monitored by light microscopy.
  • In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml), necrostatin-1 (30 μM) or caspase-8 inhibitor (50 μM ).
  • Cell Viability of organoids was analyzed indirectly by quantification of relative ATP level with the CellTiter-Glo assay fromaccording to the manufacturer's instructions.
  • Luminescence was measured on the microplate reader infinite M200 .

Read more

Crypt isolation and culture

  • Organoid culture was conducted as previously described. Crypts were released from small intestine tissue fragments by incubation for 30 min at 4°C in 2mM EDTA-PBS solution.
  • Isolated crypts were mixed with 50 μl of Matrigel , seeded in 24-well plates and 500 μl of crypt culture medium (MEM/F12 containing 50 ng/ml EGF , 100 ng/ml Noggin and 600 ng/ml-spondin 1 was added.
  • Organoids were maintained in a 37 °C humidified atmosphere under 5% CO2.

Transcription and secretion of BMP2 and FGF2 were increased in hADMPCs exposed to oxidative stress.

Recombinant murine Noggin
  • PC12 cells were cultured in CM-BSO , or CM-BSO added with recombinant murine Noggin (200 ng/mL).

Treatment of AECs with BMP2, 4, 7, noggin and gremlin

  • Freshly isolated AECs were seeded onto Oris 96 well migration plates in SCM.
  • At day 2, medium was replaced with SFM.
  • After 24 hr, supernatant was removed and fresh SFM was added containing appropriate concentrations of recombinant BMP2, 4 and 7 recombinant noggin (ocky , , ) or recombinant gremlin .

Organoid culture

  • SI of mice (creb;villinCRE and control littermates; between 3 and 5 weeks old) were isolated, opened longitudinally and cleaned in phosphate-buffered saline (PBS) supplemented with 0.1 mg/ml Nystatin and 0.1 mg/ml Gentamicin (PBS-GN).
  • The isolated intestines were incubated two times 15 min in PBS-GN on a shaker at 4 °C, followed by incubation in PBS containing 2 mℳ EDTA for 30 min at 4 °C.
  • Then the tissues were shaken vigorously in 20 ml cold PBS, of which the supernatant was discarded.
  • The tissues were transferred to 10 ml of cold PBS and shaken vigorously again to obtain fraction 2.
  • This step was repeated to obtain fraction 3.
  • Fractions 2 and 3 were combined and spun down at 200 × g for 3 min after which the supernatant, containing single cells, was removed.
  • The pellet was resuspended in PBS and filtered through a 40-μm filter.
  • The required amount was spun down at 400 × g for 6 min.
  • Five hundred or 1000 viable crypts were seeded in…
  • SI of mice (creb;villinCRE and control littermates; between 3 and 5 weeks old) were isolated, opened longitudinally and cleaned in phosphate-buffered saline (PBS) supplemented with 0.1 mg/ml Nystatin and 0.1 mg/ml Gentamicin (PBS-GN).
  • The isolated intestines were incubated two times 15 min in PBS-GN on a shaker at 4 °C, followed by incubation in PBS containing 2 mℳ EDTA for 30 min at 4 °C.
  • Then the tissues were shaken vigorously in 20 ml cold PBS, of which the supernatant was discarded.
  • The tissues were transferred to 10 ml of cold PBS and shaken vigorously again to obtain fraction 2.
  • This step was repeated to obtain fraction 3.
  • Fractions 2 and 3 were combined and spun down at 200 × g for 3 min after which the supernatant, containing single cells, was removed.
  • The pellet was resuspended in PBS and filtered through a 40-μm filter.
  • The required amount was spun down at 400 × g for 6 min.
  • Five hundred or 1000 viable crypts were seeded in 50 μl Matrigel Basement Membrane Matrix per well of a 24-wells plate.
  • The 24-wells plate was then placed at 37 °C for 30 min to allow polymerization.
  • Control crypts were cultured in crypt culture medium consisting of ulbecco's Modified Eagle Medium nutrient mixture F-12 (MEM/F12;) supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin and 100 U/ml streptomycin, 1 ng/ml recombinant mouse basic-FGF (eBioscience, San iego, CA, ), 2 ng/ml murine EGF , 1 ng/ml recombinant mouse-spondin-1 (& System , , ), 0.1 ng/ml murine Noggin , B-27 Supplement Minus AO .
  • Half of the crypts of each mouse group were cultured in crypt culture medium supplemented with 0.1 μg/ml 4OHT.
  • Medium was changed every 2–3 days, and after 7 days, the cells were isolated by incubation with 50 μl of Accumax for 30 min at 37 °C.

Read more

Crypt isolation, Enteroid Culture and Analysis

  • The pelleted epithelium was re-suspended directly into hESC Matrigel supplemented with 100 ngmL−1 recombinant mouse Noggin , 500 ngmL−1 recombinant human-Spondin , 50ngmL−1 recombinant mouse EGF , 100 ngmL−1 recombinant human Wnt3a (& systems), 10 µM Y-27632 , 10 µM SB202190 , and 500 nM LY2157299 .
  • Between 50 and 200 crypt/villi units were plated in 50 uL of matrigel on a 48 well plate.
  • After allowing the matrix to polymerize for 30 min at 37°C, each well was overlaid with 500 µL of Advanced DMEM/F12 containing the supplements 1× N-2 supplement , 1× B-27 supplement minus vitamin A , 1× Glutamax , 100 µgmL−1 penicillin/streptomycin, 1 mM Hepes buffer , and 1 mM N-Acetylcysteine.
  • Growth factors were added to the media 48 hr after plating and every 72 hr following that.
  • The entire volume of media was changed 72 hr following plating and every 72 hr after that.
  • Every 1–2 weeks organoids…
  • The pelleted epithelium was re-suspended directly into hESC Matrigel supplemented with 100 ngmL−1 recombinant mouse Noggin , 500 ngmL−1 recombinant human-Spondin , 50ngmL−1 recombinant mouse EGF , 100 ngmL−1 recombinant human Wnt3a (& systems), 10 µM Y-27632 , 10 µM SB202190 , and 500 nM LY2157299 .
  • Between 50 and 200 crypt/villi units were plated in 50 uL of matrigel on a 48 well plate.
  • After allowing the matrix to polymerize for 30 min at 37°C, each well was overlaid with 500 µL of Advanced DMEM/F12 containing the supplements 1× N-2 supplement , 1× B-27 supplement minus vitamin A , 1× Glutamax , 100 µgmL−1 penicillin/streptomycin, 1 mM Hepes buffer , and 1 mM N-Acetylcysteine.
  • Growth factors were added to the media 48 hr after plating and every 72 hr following that.
  • The entire volume of media was changed 72 hr following plating and every 72 hr after that.
  • Every 1–2 weeks organoids were passaged at a 1∶5 ratio by mechanical dissociation, pelleting, and re-plating the pellet, into new Matrigel containing the growth supplements described above.

Read more