• Purity

  >97%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ribonucleolytic activity toward yeast tRNA. Lee and Vallee (1989) Biochem. Biophys. Res. Commun.     161:121. Angiogenin produces a delta Abs    ₂₆₀/μg >1.0 in two hours, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Gln25-Pro147

• Accession #

• N-terminal Sequence    
  Analysis

  Gln25

• Predicted Molecular Mass

  14 kDa

• SDS-PAGE

  14 kDa, reducing conditions

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

Assay Procedure

Materials

(Note: prepare all reagents with RNAse-free deionized water)

• Recombinant Human Angiogenin (rhAngiogenin) (Catalog # 265-AN/CF)

• 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

1. Reconstitute bottled rhAngiogenin to 100 µg/mL with Angiogenin Dilution Buffer.

2. 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.

3. 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

4. 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.

5. Incubate tubes at 37 °C for two hours.

6. Dilute perchloric acid to 6% (v/v) in RNAse-free deionized water. Place on ice.

7. After incubation, add 200 µL cold 6% perchloric acid to each tube. Mix well and incubate on ice for at least 10 minutes.

8. Centrifuge tubes at 2-8 °C at 13,000 rpm for 20 minutes.

9. Add 200 µL RNAse-free water to a third set of tubes and place on ice.

10. Carefully transfer 100 µL of supernatant from each sample to a tube of RNAse-free water prepared in step 9. Mix well.

11. Load 200 µL from each tube into a UV plate. Load reaction blanks, followed by low to high concentrations of rhAngiogenin.

12. Fill cuvette with RNAse-free water and place in cuvette port (required for path length correction).

13. Read the plate at 260 nm in endpoint mode.

14. 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 = [Abs_sample - Abs_blank] x 6 (dil. factor)

15. The rhAngiogenin activity is equal to the slope of the plot ( delta Abs₂₆₀/µg rhAngiogenin).

Per Reaction:

• rhAngiogenin: 2.00, 1.330, 0.889, 0.593, 0.395, 0.263, 0 µg

• tRNA: 60 units/mL

Background: Angiogenin

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.

• Entrez Gene IDs:

  283 (Human)

• Alternate Names:

  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