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Tuesday, February 28, 2012

Horse Radish Peroxidase (HRP) Mechanism of Action

Horse Radish Peroxidase(HRP) is heme-containing protein isolated from wild horse radish roots.HRP catalyzes the transfer of two electrons from a substrate to hydrogen peroxide(H2O2), to generate H2O and an oxidized donor. When 3',3'-diaminobenzidine(DAB) is used as a substrate, the oxidized product polymerizes to form a brown precipitate in water and ethanol. In presence of cobalt or nickel the product will be blue-black. This form the basis of chromogenic assays for peroxidase in immunochemistry, western blotting etc.

Horse Radish Peroxidase Mechanism - Developing a western blot / Elisa

Developing a western blot:
Developing an immunoblot of HRP conjugated antibody is explained here, developing solution is made by dissolving the substrate 0.03% diaminobenzidene(DAB) in PBS and 1% CoCl2,   to this 0.1% hydrogen peroxide (H2O2)is added prior to the incubation with the membrane. even preformulated DAB solutions are readily available. Wash the membrane with wash buffer and incubate with developing solution for 30 seconds to one minute,DAB reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to the target protein. The reaction can be stopped by adding water.

Saturday, February 25, 2012

Normal Phase & Reverse Phase Chromatography

Normal Phase
In a very non-polar environment, hydrophilic molecules will tend to associate with each other (like water drops on an oily surface). The hydrophilic molecules in the mobile phase will tend to adsorb to the surface on the inside and outside of a particle if that surface is also hydrophilic. Increasing the polarity of the mobile phase will subsequently decrease the adsorption and ultimately cause the sample molecules to exit the column. This mechanism is called Normal Phase Chromatography. It is a very powerful technique that often requires non-polar solvents. Due to safety and environmental concerns this mode is used mostly as an analytical technique and not for process applications.

Reversed Phase
The opposite of normal phase, or Reversed Phase Chromatography, results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase. Decreasing the mobile phase polarity by adding more organic solvent reduces the hydrophobic interaction between the solute and the solid support resulting in de-sorption. The more hydrophobic the molecule the more time it will spend on the solid support and the higher the concentration of organic solvent that is required to promote de-sorption.

Reversed phase chromatography (RPC) is the most popular separation technique at analytical scale, because:
RPC applies to a very wide range of molecules including charged and polar molecules,
RPC allows precise control of variables such as organic solvent type and concentration, pH, and temperature,
RPC columns are efficient and stable,
RPC is a robust technique.

At process scale, RPC is not typically used for protein purification due to the presence of the organic solvent which can cause denaturation of proteins and destroys their biological activity. Reversed phase chromatography is the most dominant analytical HPLC technique and there are many different stationary phases available for method optimization.

Tosoh Bioscience Resource Centre

Friday, February 3, 2012

Plasmid Extraction / plasmid prep protocol & principle

Plasmids are double stranded, circular, extra chromosomal, self replicating DNA molecules which carry additional genes which helps the bacteria to survive in extreme conditions of stress.

Many methods have been developed to purify plasmids, these methods involves these steps
  • Growth of Bacterial Culture.
  • Harvesting and Lysis of Bacterial Cells.
  • Purification of Plasmid DNA.

Bacterial Plasmid - Isolation / Extraction
Bacterial Plasmid - Image Source

Growth of Bacterial Culture:

overnight culture of bacterial cells having plasmid can be used for plasmid extraction/ plasmid prep. The volume of bacterial culture used depends on the amount of plasmid required. if the plasmid has any markers like antibiotic genes, suitable antibiotic can be used which ensures only plasmid containing bacteria grows in it, why it is because bacterial cells tend to loose plasmid sometimes.

Harvesting and Lysis of Bacterial Cells:

Once the culture attains particular OD, depends on the amount you are going to start with, bacterial cells can be harvested by centrifugation. After centrifugation, Cells will settle at the bottom which can be collected and lysed for plasmid extraction.

Purification of Plasmid DNA:

Plasmid can be purified using silica columns.

Reagents Required:

Solution I

(Tris pH 8.0, EDTA- Ethylene diamine tetra acetic acid and Glucose)


Lysozyme- optional

50mM glucose
25mM Tris-Cl (pH 8.0)
10mM EDTA (pH 8.0)

Prepare Solution I from standards stocks of 100ml and autoclave for 15mins.

Solution II

0.2N Sodium hydroxide (Freshly diluted from 10N NoaH Stock)
1% SDS- Sodium dodecyl sulphate

(Alkaline pH 12.0)

Solution III
5M Potassium acetate – 60ml
Glacial Acetic acid – 11.5ml
Water – 28.5ml
(Acidic pH 5.4)

Plasmid Extraction / plasmid prep Principle

Plasmid prep process

Image Source: Favorgen Biotech Corp

Solution I
Solution I has EDTA in it which chelates magnesium and calcium ions, there by destabilizing the membrane. Glucose helps to maintain the osmolarity. Lysozyme which can be used to lyse the membrane.

Solution II
NaOH which is highly alkaline, SDS which creates pores on the cell wall and NaOH helps in loosening the cell wall, non-supercoiled DNA gets denatured at higher pH (due to NaOH), hydrogen bonding in DNA molecule is broken down and the two strands separates, but supercoiled plasmid DNA will be intact.

Solution III
Sodium or potassium acetate , when the acidic solution is added the separated strands reaggregates and forms a tangled mass, on centrifugation this insoluble network gets pelleted and the plasmid DNA will be left out in supernatant.

Protocol: Plasmid Extraction from E.coli

1. Transfer 1.5ml of bacterial culture to a micro centrifuge tube.

2. Pellet cells by centrifuging at 12,000 rpm for 2 minutes

3. CAREFULLY remove the supernatant.

4. Add 250 ul of Solution I /RNAase.

5. Resuspend the pellet by vortexing briefly or by pipetting up and down.

6. Add 250 ul of Solution II

7. Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex!!!

8. Leave at room temperature for 5 minutes but NOT MORE than 5 minutes.

9. Add 350 ul of Solution III

10. Mix by inverting the tube 6-8 times

11. Centrifuge at 12,000 rpm for 5 min

12. While your tubes are spinning place a Spin column in a 2-ml collection tube.

13. After centrifugation, CAREFULLY transfer the supernatant to the column. DO NOT disturb the pellet.

14. Centrifuge the column and collection tube at 12,000 rpm for 1 min.

15. Discard the flow through collected in the collection tube.

16. Replace the column in the collection tube

17. Add 250 ul of 70% ethanol into the column and centrifuge for 1 minute at 12,000 rpm, discard the flow through, reapeat step 17

18. Place the column in a sterile 1.5ml tube and leave in room temperature for 2 mins.

19. Add 50ul of deionised water or TE buffer into the column.

20. Centrifuge at 12,000 rpm for 1 min.

21. Discard the column. Plasmid DNA is in the deionised water or TE buffer will be collected in the 1.5ml tube.

22. Place the tube containing plasmid DNA on ice for further use or store at –20oC.

DNA concentration can be checked using nanodrop spectrophotometer, and the analysis of the extracted plamsid can be done by running on an agarose gel.

There are various plasmid preparation / plasmid prep kits which can be used to extract the DNA like QIAGEN Plasmid Extraction kit, Promega Plasmid Extraction kit, Plamid isolation kit from GenScript , etc

Plasmid Extraction Kit
Qiagen Plasmid Extraction Kit - Image Source: Qiagen


J. Sambrook, Molecular Cloning 3rd Edition

TA Brown, Gene Cloning & DNA Analysis

QIAGEN Plasmid Extraction Technical Notes

Internet Sources