Measured by its ribonucleolytic activity toward yeast tRNA. Lee and Vallee (1989) Biochem. Biophys. Res. Commun. 161:121. Angiogenin produces a delta Abs260/μg >1.0 in two hours, as measured under the described conditions.
Source
E. coli-derived human Angiogenin protein Gln25-Pro147
>97%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Bioactivity
Theoretical MW
14 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
14 kDa, reducing conditions
Publications
Read Publications using 265-AN in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -20 to -70 °C as supplied.
3 months, -20 to -70 °C under sterile conditions after reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein.
Purity
>97%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 10 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.
Assay Procedure
(Note: prepare all reagents with RNAse-free deionized water)
Recombinant Human Angiogenin (rhAngiogenin) (Catalog # 265-AN)
Angiogenin Dilution Buffer: PBS, 0.1% BSA
Standard Curve Buffer: 30 mM HEPES, 0.1% BSA, pH 7.0
0.3 M Hepes, 0.3 M NaCl, pH 7.0
BSA, 0.1% in deionized water
tRNA, Type X (Sigma, Catalog # R9001), 600 units/mL in Standard Curve Buffer
RNAse-free water (Ambion cat # AM9920, DEPC treated water)
Perchloric Acid (Fisher, Catalog # A469-250)
96 well clear UV-transparent microplate (Corning, Catalog # 3635)
UV transparent Spectophotometer cuvette
Plate Reader (Model: Spectramax Plus by Molecular Devices) or equivalent
Reconstitute bottled rhAngiogenin to 100 µg/mL with Angiogenin Dilution Buffer.
Dilute the 100 µg/mL rhAngiogenin to 40, 26.7, 17.8, 11.9, 7.9 and 5.26 µg/mL with Standard Curve Buffer. Include two blank controls.
Prepare reagent mix using the following volumes for each one tube tested. Prepare at least one extra tube to accommodate volumetric recovery loss (ie. prepare 9x for this protocol: six dilutions and two blanks per lot of rhAngiogenin tested): 0.3 M Hepes, 0.3 M NaCl, pH 7.0 1x=20 µL 0.1% BSA 1x=20 µL tRNA, 600 units/mL 1x=20 µL RNAse-free deionized water 1x=90 µL
Combine 150 µL of reagent mix and add 50 µL of each rhAngiogenin dilution to the appropriate tubes. As controls, combine 150 µL of reagent mix with 50 µL of Angiogenin dilution buffer. Mix well.
Incubate tubes at 37 °C for two hours.
Dilute perchloric acid to 6% (v/v) in RNAse-free deionized water. Place on ice.
After incubation, add 200 µL cold 6% perchloric acid to each tube. Mix well and incubate on ice for at least 10 minutes.
Centrifuge tubes at 2-8 °C at 13,000 rpm for 20 minutes.
Add 200 µL RNAse-free water to a third set of tubes and place on ice.
Carefully transfer 100 µL of supernatant from each sample to a tube of RNAse-free water prepared in step 9. Mix well.
Load 200 µL from each tube into a UV plate. Load reaction blanks, followed by low to high concentrations of rhAngiogenin.
Fill cuvette with RNAse-free water and place in cuvette port (required for path length correction).
Read the plate at 260 nm in endpoint mode.
Plot a linear curve with the amount of rhAngiogenin (µg per well) on the x-axis, and the adjusted Abs260 on the y-axis: Adjusted Abs260 = [Abssample - Absblank] x 6 (dil. factor)
The rhAngiogenin activity is equal to the slope of the plot ( delta Abs260/µg rhAngiogenin).
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Angiogenin Protein
ALS9
ANG
Angiogenin
angiogenin, ribonuclease, RNase A family, 5
EC 3.1.27
EC 3.1.27.-
epididymis luminal protein 168
HEL168
MGC22466
MGC71966
Ribonuclease 5
RNase 5
RNASE5RNASE4
Background
Angiogenin was initially purified from serum-free media conditioned by growth of a human adenocarcinoma cell line HT-29 based on its ability to initiate vascularization in the chicken embryo chorioallantoic membrane. A number of other tumor, as well as normal, cell lines can also secrete Angiogenin. In addition, Angiogenin is present in normal human plasma at levels as high as 60-120 ng/mL. Unlike other angiogenic factors such as FGF, Angiogenin is neither mitogenic nor chemotactic for vascular endothelial cells in vitro. However, Angiogenin can stimulate capillary and umbilical vein endothelial cells to produce diacylglycerol and secrete prostacyclin by phospholipase activation. Angiogenin, absorbed on plastic, can also support endothelial and fibroblast cell adhesion and spreading.
Surprisingly, Angiogenin has been found to be a member of the ribonuclease superfamily with approximately 35% sequence similarity at the amino acid level with pancreatic RNase. Angiogenin exhibits ribonucleolytic activity that is distinctly different than that of pancreatic RNase A. The ribonucleolytic activity of Angiogenin toward most RNase A substrates is much lower than that of RNase A. Nevertheless, the ribonucleolytic activity of Angiogenin is essential to its angiogenic activity since inhibition of the Angiogenin RNase activity will also abolish angiogenesis activity. Similar to several members of the RNase superfamily, Angiogenin is a cytotoxic agent that can abolish cellular protein synthesis. It has been demonstrated that Angiogenin-dependent protein synthesis inhibition can be attributed to the function of Angiogenin as a cytotoxic tRNA-specific RNAase.
A cell-surface Angiogenin binding protein has been purified and characterized. Tryptic peptide mapping and sequence analysis indicate that this binding protein is a member of the actin family.
The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.
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