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Protein Purification

APPLICATION overview, CHALLENGES AND SOLUTION

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Protein Purification
dsDNases
M-SAN HQ
M-SAN HQ ELISA kit
SAN HQ
SAN ELISA kit
IsoPol BST+
HL-SAN
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Endonucleases Non-Specific
HL-SAN
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Application Overview

Protein purification is a fundamental step in various biotechnological applications. However, the presence of nucleic acids, particularly genomic DNA, can interfere with the purification process and downstream applications.

HL-SAN offers a specialized solution for DNA digestion, facilitating a more efficient protein purification workflow.

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Protein Purification
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No items found.
M-SAN HQ
M-SAN HQ ELISA kit
SAN HQ
SAN HQ ELISA kit
SAN HQ GMP
SAN HQ
dsDNases
Heat&Run gDNA removal kit
For Detailed Info Including:
  • Product overview
  • Performance data & figures
  • Specifications
  • Documents
  • FAQs
  • Ordering Info
  • Protocols
  • Publications

Application Overview

Protein purification is a fundamental step in various biotechnological applications. However, the presence of nucleic acids, particularly genomic DNA, can interfere with the purification process and downstream applications.

HL-SAN offers a specialized solution for DNA digestion, facilitating a more efficient protein purification workflow.

THE PROBLEM These ENZYMEs SOLVE

Host-cell DNA can be problematic in the purification of DNA-binding proteins. They not only interfere with the purification process but also affect downstream analyses and applications. Traditional nucleases used for DNA removal are often difficult to inactivate, posing a risk of residual activity that can interfere with subsequent steps.

THE PROBLEM These ENZYME SOLVES

Host-cell DNA can be problematic in the purification of DNA-binding proteins. They not only interfere with the purification process but also affect downstream analyses and applications. Traditional nucleases used for DNA removal are often difficult to inactivate, posing a risk of residual activity that can interfere with subsequent steps.

THE PROBLEM These ENZYMes SOLVE

Host-cell DNA can be problematic in the purification of DNA-binding proteins. They not only interfere with the purification process but also affect downstream analyses and applications. Traditional nucleases used for DNA removal are often difficult to inactivate, posing a risk of residual activity that can interfere with subsequent steps.

Fig .
Fig .

The Solution

HL-SAN addresses these challenges effectively. It can be easily inactivated by treatment with a reducing agent, and its high isoelectric point (pI 9.6) allows for easy separation from most target proteins. Moreover, HL-SAN is optimized for high salinity conditions and is resistant to non-ionic detergents, making it ideal for dissociating DNA from DNA-protein complexes. These features collectively make HL-SAN the superior choice for DNA digestion in protein purification workflows.

Download Tech note:  HL-SAN for DNA removal in protein purification

Guidelines for DNA removal

The amount of HL-SAN needed for DNA removal from cell extracts or lysates depends on several factors; expression strain, properties of the target protein, buffer composition, temperature, etc. The following is therefore considered as guidelines: Add 1000 U HL-SAN per ml sample with 0.3–0.75 M NaCl and incubate at 15–37°C for 30–60 minutes or at 4°C overnight. Mg2+ is a necessary cofactor for activity.

DNA may cause problems both during purification and if present in the final product. In the initial stages of purification, fragmentation of genomic DNA in the lysate/extract is usually sufficient to significantly improve results. However, even small amounts of DNA can cause issues if the end product is to be used in highly sensitive molecular assays. In the latter case, it is recommended to implement an additional decontamination step further downstream.

RECOMMENDED OPERATING CONDITIONS
Condition
Optimal
Effective*
Salt (NaCl/KCl)
500 mM
50 mM-1 M
Temperature
~35°C
4-50°C
Mg2+/Mn2+
5-20 mM
1-40 mM
pH
9.0
7.0-9.5

*Effective is defined as the condition which HL-SAN has ≥ 10 % of its activity as compared to optimal conditions.

Inactivation

Inactivation is achieved by adding reducing agents like TCEP or DTT, where TCEP* is the recommended reducing agent. The inactivation protocol can be adapted to several workflows by varying incubation time, temperature and concentration of the reducing agent. In general >99% inactivation is achieved after 5–10 minutes at 25–37°C.

To avoid reactivation, maintain a low concentration of reducing agent, 0.1–0.5 mM DTT or TCEP, or use prolonged incubation times with 10–20 mM TCEP upon inactivation.

*Low stability in phosphate buffers, especially at neutral pH.

GUIDELINES FOR INACTIVATION
Temperature/Time
DTT
TCEP
4°C/18 hours
-
10 mM
25°C/60 minutes
10 mM
5 mM
30°C/30 minutes
10 mM
5 mM
40°C/30 minutes
5 mM
1 mM
50-70°C/30 minutes
1 mM
1 mM

Removal

The very high pI (9.6) of HL-SAN results in tight binding to cationic columns. Even at pH 9.0 with 0.2M salt, less than 0.02% leakage in flow-through/wash is observed. It is not recommended to use anionic IEX columns for removal of HL-SAN as the glycosylation of HL-SAN result in column binding

CONDITIONS FOR BINDING HL-SAN TO SP-SEPHAROSE

pH
Salt
pH 7
≤ 0.3 M
pH 8
≤ 0.3 M
pH 9
≤ 0.2 M
Fig 1. HL-SAN binds tightly to SP-sepharose columns at pH 9.0 with 0.2M salt (less than 0.02% leakage).

DNA removal from various samples

Sample
HL-SAN final concentration
Recommended conditions
DNA removal*
Decontamination**
Protein
100 U/ml
1000 U/ml
30 minutes at 25–37°C
Reagent
100 U/ml
1000 U/ml
30 minutes at 25–37°C
Cell extract
1000 U/ml
N/A
60 minutes at 25–37°C or 4°C overnight
Cell lysate (soluble fraction)
500 U/ml
N/A
60 minutes at 25–37°C or 4°C overnight
Viscosity reduction
25-50 U/ml
10-20 minutes at 25°C

* DNA amount is reduced to a level that cannot be detected by visualization using agarose gel electrophoresis.

** DNA amount is reduced to a level generally not detectable by a 23S rDNA qPCR assay.

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Application Background

See complete product details  for

M-SAN HQ
M-SAN HQ ELISA kit
SAN HQ
SAN HQ ELISA kit
SAN HQ
Endonucleases Non-Specific