Citations & References

Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of “masked” proteins

  • Authors: H. Hilz et al.
  • Journal: Eur J Biochem (1975) 56(1):103-108

Product FAQ

Can I use Thermo Scientific 2X RNA Loading Dye for native gel electrophoresis?

Answer

Thermo Scientific 2X RNA Loading Dye contains 95% formamide as a denaturing agent and hence cannot be used for native gel electrophoresis.

Answer Id: E8867

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Product FAQ

I am using your ZOOM IPGRunner system. What is the sample rehydration buffer?

Answer

The sample rehydration buffer, also known as the sample buffer, is used to denature and solubilize protein samples, and rehydrate the IPG strips prior to performing IEF using the ZOOM IPGRunner system. Due to the large variety of proteins, there is no universal sample rehydration buffer. The sample rehydration buffer must maintain proteins in solution during rehydration of the IPG strips and IEF, and must not have any effect on the pI of the protein. The buffer typically contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT).

Note: To obtain high resolution, IEF is usually performed under denaturing and reducing conditions. Use a denaturing sample rehydration buffer for sample preparation (see manual for recipe).

Answer Id: E10672

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Product FAQ

How can I perform RNA electrophoresis?

Answer

For nondenaturing RNA electrophoresis, we recommend using our E-Gel Precast Agarose Gels. Please note that E-Gel Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.

For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.

For denaturing RNA electrophoresis, our E-Gel EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:

RNA Loading Buffer:
Deionized formamide: 200 µL
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10 mM EDTA): 40 µL
Deionized formaldehyde: 76 µL
Water: 14 µL

Denaturing Electrophoresis Conditions:
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel EX agarose gel.
5. Electrophorese for 30 minutes.

For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.

Answer Id: E7957

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Product FAQ

Which sample buffer do you recommend for IEF fractionation?

Answer

Proper sample preparation is the key to the success of IEF fractionation. An ideal sample buffer must maintain the proteins in solution during IEF and not have any effect on the pI of the protein. The sample buffer generally contains a denaturing agent (urea or urea/thiourea), solubilizing agent (non-ionic or zwitterionic detergent and ampholytes), and reducing agent (DTT). Due to the large variety of proteins present in different cells and tissues, it is not possible to have a sample preparation protocol that is suitable for all proteins. Based on the starting material and goal of the experiment, the sample preparation protocol needs to be determined empirically. Please refer to the manual (http://tools.thermofisher.com/content/sfs/manuals/zoomieffractionator_man.pdf) for specific sample preparation guidelines.

Answer Id: E10661

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Product FAQ

What are the recommended conditions for proteinase K treatment when isolating RNA or DNA samples?

Answer

Concentration: Generally proteinase K is used in the concentration range of 50 to 500 µg/mL at 65 degrees C in the presence of SDS (0.5-1%).

Temperature optimum: 65 degrees C; 12X more active at 65 degrees C than at 25 degrees C.

pH: Proteinase K is stable over a wide pH range (4.0 to 12.5), with optimal activity at pH 6.5 to 9.5. It is most stable at pH 8.
Inactivation: Heat inactivate Proteinase K at 80 degrees C for 15 min. Phenol extraction is the recommended method to ensure complete inactivation.

Inhibited by: PMSF (0.1 to 1.0 mM of PMSF is usually sufficient for inhibition of Proteinase K)

Not inhibited by: Proteinase K is not inactivated by metal ions, chelating agents (e.g., EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. Activity can be stimulated by addition of denaturing agents (SDS and urea).

Answer Id: E2960

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Product FAQ

How can I dissociate my biotinylated molecule from Dynabeads Streptavidin magnetic beads?

Answer

The streptavidin-biotin interaction is the strongest known non-covalent biological interaction between a protein and a ligand. Binding of biotin and streptavidin is very rapid and, once formed, the complex is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Normally, very harsh methods are required to dissociate the biotin from streptavidin, which will be irreversibly denatured by the procedure. Biotin-streptavidin interactions are more easily reversible when biotin derivatives with lower binding affinity are used along with chemically modified streptavidins with lower biotin binding affinity. When modified biotins and streptavidins are used, gentle methods for inducing reversible binding are available

To dissociate biotinylated proteins from streptavidin, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 mins or incubate them in 8 M guanidinium hydrochloride, pH 1.5.

Answer Id: E13034

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Product FAQ

How can I dissociate my biotinylated molecule from Dynabeads Streptavidin magnetic beads?

Answer

The streptavidin-biotin interaction is the strongest known noncovalent biological interaction between a protein and other molecule. The bond formation between biotin and streptavidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment, requiring a specific form and a normally gentle way to dissociate biotin from streptavidin, very harsh methods are often required to dissociate the biotin from streptavidin, which will denature the streptavidin. A couple of these methods are discussed below:

Biotinylated nucleic acids: To dissociate biotinylated nucleic acids from Dynabeads Streptavidin magnetic beads, incubate the beads in 95% formamide + 10 mM EDTA, pH 8.2, for 5 minutes at 65°C or for 2 minutes at 90°C. Pull the beads to the tube wall with the magnet and remove the supernatant containing the biotinylated nucleic acid from the tube. Holmberg et al. (Electrophoresis 26, 501-510, 2005), report release of biotinylated DNA from streptavidin beads after short incubation in deionized H2O (though we have not tested this).

Biotinylated proteins: For biotinylated proteins, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 min.

Answer Id: E7935

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Product FAQ

Do you have a protocol for RNA electrophoresis on your E-Gel Agarose Gels?

Answer

E-Gel EX Agarose Gels can be used for either DNA or RNA. RNA separation occurs under nondenaturing or denaturing conditions. Please note, our gels are not QC tested for the presence of RNases. See our suggestions below for running your nondenaturing or denaturing samples:

Nondenaturing Conditions
1. Mix RNA sample with 15 µL of RNase-free water.
2. Do not heat. Load the entire sample onto the E-Gel EX Agarose Gel.
3. Electrophorese for 30 minutes.

Denaturing Conditions*
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65°C for 10 minutes to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto E-Gel EX Agarose Gel.
5. Electrophorese for 30 minutes.

*The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-95%. Heating the sample for 5 minutes at 65°C should be sufficient for denaturing. Using other denaturing agents like glyoxal, formaldehyde, or urea will result in very poor separation and band morphology on E-Gel EX Agarose Gels. Additionally, we do not recommend running samples with RNA loading buffer on the same gel as samples loaded with water.

Answer Id: E7965

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Product Literature

Application Note: Environmental Water Applications Notebook: Hexavalent Chromium - Metals

Citations & References

A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation.

  • Authors: Wondrak EM, Nashed NT, Haber MT, Jerina DM, Louis JM
  • Journal: J Biol Chem (1996) 271:4477-4481
  • PubMed ID: 8626801
Catalog #
  • H2930(Discontinued)

Manual / Product Insert

User Guide: E-Gel Technical Guide

Version: Rev. A (August 2, 2014)
Catalog #
  • A25798
  • G700801
  • G700802
  • G720802
  • G800801
  • G800802
  • G800804
  • A25798ST(Discontinued)
  • G6511ST(Discontinued)
  • G6512ST(Discontinued)
  • G661002(Discontinued)
  • G6612ST(Discontinued)
  • G661808(Discontinued)
  • A25798EU-LSG
  • A25798UK-LSG
  • G6511EU-LSG
  • G6511UK-LSG
  • G6512EU-LSG
  • G6512UK-LSG
  • G6612EU-LSG
  • G6612UK-LSG

Product Literature

SMART Digest Peptide Mapping and Quantitation Compendium

Manual / Product Insert

ZOOM IPGRunner System

Version: 15 February 2012