as fic
ld nti
So ie
w Sc
No mo
er
Th
RiboLock™ Ribonuclease Inhibitor
Efficient RNA Protection
•Rapid and complete protection of RNA
•Free of DNases, RNases and phosphatases
P25
Part of Thermo Fisher Scientific
Rapid and Complete RNA Protection
Fermentas RiboLock™ Ribonuclease Inhibitor is a mammalian protein of non-human origin that completely protects RNA in
molecular biology applications. The protein “locks” onto RNases and thus prevents RNA degradation.
The Fermentas RiboLock™ Ribonuclease Inhibitor (RI) is purified from recombinant E.coli.
Features
• Inhibits eukaryotic RNases, including RNase A,
RNase B and RNase C
• Rapid and complete protection of RNA
• Inhibits RNases at temperature up to 55°C
• Active within the pH 5-9 range
• Free of DNases, RNases and phosphatases
• Compatible with all molecular biology procedures
Applications
• Inhibitions of RNases in the following applications:
–IIdentification of specific ribonuclease activities
–cDNA synthesis
–In vitro transcription and translation
• Isolation of eukaryotic mRNA
RiboLock™ Ribonuclease Inhibitor Increases RT-PCR Sensitivity
Without
With
RiboLock™ RI RiboLock™ RI
µg total RNA template
M
1.0 0.3 0.1 1.0 0.3 0.1
3 kb
Decreasing amounts of mouse heart total RNA was used as a template for reverse transcriptase in the
absence or presence of RiboLock™ Ribonuclease Inhibitor.
cDNA was amplified by PCR using High Fidelity PCR Enzyme Mix (#K0191) and primers specific to the
dystrophin gene.
M 1 2 3 4 5 6
M –GeneRuler™ 1 kb DNA Ladder, ready-to-use (#SM0311)
1-3–mouse heart total RNA without RiboLock™ Ribonuclease Inhibitor
4-6–mouse heart total RNA with RiboLock™ Ribonuclease Inhibitor
Efficiency of RNA protection, %
RiboLock™ Ribonuclease Inhibitor Provides Superior Protection from RNase A
100
90
80
pre-incubation
in-assay
For the “in-assay”, aliquots (0.5 ml) of total yeast RNA (2.5 mg) were
supplemented with 8 units of either RiboLock™ Ribonuclease Inhibitor
or a broad specificity ribonuclease inhibitor from another vendor and
incubated for 5 minutes at 37°C in the presence of 5 ng of RNases A.
70
60
50
40
30
For the “pre-incubation assay”, the ribonuclease inhibitors were mixed
with RNase A and incubated for 15 min at 22°C. These pre-incubated
solutions were added to 0.5 ml aliquots of the total yeast RNA (2.5 mg).
The mixtures were then incubated for 5 minutes at 37°C.
20
10
0
Two assays were performed to assess the effectiveness of RNA protection.
Without RI
RiboLock™ RI Broad specificity RI
from another vendor
Aliquots (0.5 ml) of 10% TCA were added to the mixtures to terminate
reactions and precipitate large RNA molecules. The amount of large RNA
was determined spectrophotometrically at 260 nm.
RiboLock™ Ribonuclease Inhibitor protects RNA rapidly and completely!
www.thermoscientific.com/fermentas
Efficient RNA Protection
Complete Inhibition of RNase A by RiboLock™ Ribonuclease Inhibitor
20 u RiboLock™ Ribonuclease Inhibitor
RNase A
pg
pg
pg ng
pg
2
125 125 250 500
Aliquots (20 µl) of total human RNA (1 µg) were supplemented at 37°C with 20 u of the
RiboLock™ Ribonuclease Inhibitor and increasing amounts of RNase A.
M –RiboRuler™ High Range RNA Ladder, ready-to-use, (#SM1823)
C –total human RNA
1 –total human RNA with RNase A
2-5–total human RNA with RiboLock™ Ribonuclease Inhibitor and RNase A
M C 1 2 3 4 5
RiboLock™ Ribonuclease Inhibitor efficiently protects RNA and inhibits up to 2 ng /20 µl of RNase A.
RiboLock™ Ribonuclease Inhibitor Exhibits High Thermostability
20 u RiboLock™ Ribonuclease Inhibitor
50 pg RNase A
C
37°
C
37°
C
42°
C
45°
C
50°
C
55°
Aliquots (20 µl) of total human RNA (1µg) were supplemented with 50 u of the RiboLock™
Ribonuclease Inhibitor and 50 pg of RNase A and incubated at increasing temperatures.
M C 1 2 3 4 5 6
M –RiboRuler™ High Range RNA Ladder, ready-to-use, (#SM1823)
C –total human RNA
1 –total human RNA with RNase A
2-6–total human RNA with RiboLock™ Ribonuclease Inhibitor and RNase A
The RiboLock™ Ribonuclease Inhibitor is active up to 55°C! The high thermostability of RiboLock™
Ribonuclease Inhibitor is important for successful reverse transcription reactions when secondary
structure of RNA necessitates higher incubation temperatures.
www.thermoscientific.com/fermentas
PureExtreme® Quality & Performance
Quality & Performance
Production in class D clean room facilities and Fermentas’ stringent quality control with the most
advanced tests guarantees the purity of our products. ISO9001, ISO14001 and ISO13485 assures
product quality consistency and lot-to-lot reproducibility.
RiboLock™ Ribonuclease Inhibitor is Free of DNases and Phosphatases
RiboLock™ Ribonuclease Inhibitor
from Fermentas
DNA used:
ss
ds
Ribonuclease Inhibitor
from another vendor
ss
DNA used:
ds
Intact DNA
Degraded DNA
Heavy DNA degradation
No DNA degradation
Conclusions: RiboLock™ Ribonuclease
Inhibitor is free of any DNase
and phosphatase contamination
Ribonuclease Inhibitor from
another vendor is DNase contaminated
Single-stranded (ss) and double-stranded (ds) radiolabeled DNA fragments were incubated 4 hours at 37°C with RiboLock™ Ribonuclease
Inhibitor and a ribonuclease inhibitor from another vendor. The samples were analyzed by PAGE and phospho-imaging.
The RiboLock™ Ribonuclease Inhibitor from Fermentas is free of contaminating activities that may
interfere with your downstream applications.
Ribonuclease Inhibitor
RNase A
Interaction of Ribonuclease Inhibitor with RNase A
The structure is from the RCSB Protein Data Bank, 1DFJ, ©RCSB
Ribonuclease inhibitor “locks” onto RNase A .
As the result, degradation of RNA by RNase A is
completely inhibited.
Product
RiboLock™ Ribonuclease Inhibitor
(40 u/µl)
Catalog #
Size
EO0381
EO0382
E00384
2500 u
4x2500 u
24x2500 u
Bulk orders and special concentrations are available upon request.
PureExtreme® is a registered trademark of Fermentas.
GeneRuler™ , RiboRuler™ and RiboLock™ are Fermentas trademarks.
www.thermoscientific.com/fermentas
Part of Thermo Fisher Scientific
Fermentas – a member of your research team
© Fermentas 2008