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Wednesday, September 25, 2013

Immunoprecipitation: Procedure, Analysis and Applications


Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.

Immunoprecipitaion in general involves the following Steps:
  • Incubate sample with antibody against protein of interest.
  • Separate antibody-protein complex from remaining sample
  • Analysis

Basic Principle Of Immunoprecipitaion

Immuno precipitation


A protein complex can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex.

Applications of Immunoprecipitation Technique

  1. Isolate / Detect Proteins of interest
  2. Enrichment of low abundant proteins
  3. Study protein-protein interaction and protein complexes
  4. Identify unknown proteins in a protein complex
  5. Verify protein expression in a specific tissue.
Immunoprecipitaion Procedure / Protocol

Immunoprecipation Procedure / protocols involves the following steps
  1. Sample Preparation
  2. Use of an Isolate control
  3. Pre-Cleaning of sample
  4. Antibody Incubation
  5. Precipitation of Protein / Protein Complex
  6. Washing
  7. Elution
  8. Analysis of the Precipitate
Lets look into each Step:

Sample Preparation

Samples used for immunoprecipitaion can be of any samples of biological origin.
Lysis Buffer: Choice of buffer depends on goal of the immunoprecipitation experiment

Lysis Buffer Criteria
Concentration Range
Type and amount of detergent
Non-Ionic : 0.1 – 2.0 %
Ionic: 0.01% to 0.5%
Amount of Salt
0-1 M
Presence of EDTA
6.0 – 9.0

Lysis Buffer should always contain protease inhibitors and phosphatise inhibitors
Store lysates at -20dc or  -80dc
Avoid freeze thaw cycles

Isotype Control
Isotype control is used to establish the specificity of the signal and the amount of non-specific background. It should be done simultaneously with the IP antibody but in a different tube.

This step is done to avoid any non-specific binding and thereby avoiding the background signal. This step helps to reduce the background and improve signal to noise ratio. It is an optional step to improve the signal.
Antibody Incubation
Amount of antibody required need to be find out and it is important to have optimal antibody to protein ratio to have better results.
Different antibody to protein concentration ratios can be tried out (1:100 to 1:1000), Antibody incubation is done by incubating the IP antibody with the lysate by gentle agitation, it can be done at room temperature for 2hrs or at 4dc overnight . Incubation time and antibody concentration need to be optimized for better results.
Precipitation of Protein / Protein Complex
Protein A,G or L coupled to beads (Aarose or Sepharose) are most commonly used for Protein precipitation. Base on the host species and the type IP antibody beads can be selected.
Antibody can be directly conjugated to the beased the advantage of this is that of having lesser non-specific bands.
Immunoprecipitation is done by incubating with the antibody and then centrifuging it at 4dc.

Washing is done to remove the non-specifically bound proteins from the immunoprecipitate. Washing is generally  done with Lysis buffer or PBS. PBS is less stringent and can be used for analysis of protein-protein complexes.

Elution step is to dissociate the specifically bound proteins from antibody-bead complex.
Elution Buffers:
2X Laemmli Buffer: Harsh Buffer can denature protein
Glycine Gradient (upto 1M):  More gentle can dissociate protein of interest without eluting IP antibody.

Analysis of the Precipitate:

Analysis can be done using the following methods
  1. SDS – PAGE
  2. Western Blotting
  3. Gel band Excision and sequencing
  4. Mass Spectrometry
  5. Scintillation counter or X-ray film for radioactive samples, etc.


Abcam Technical Resources
Thermo-Scientific Technical Resources 

Friday, September 20, 2013

Sequence Alignment and Primer Designing Using Bioedit

BioEdit is a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7. An intuitive multiple document interface with convenient features makes alignment and manipulation of sequences relatively easy on your desktop computer. Several sequence manipulation and analysis options and links to external analysis programs facilitate a working environment which allows you to view and manipulate sequences with simple point-and-click operations.

BioEdit's features include:
  • Several modes of hand alignment
  • Automated ClustalW alignment
  • Automated Blast searches (local and WWW)
  • Plasmid drawing and annotation
  • Accessory application configuration
  • Restriction mapping 
  • RNA comparative analysis tools
  • Graphical matrix data viewing tools
  • Shaded alignment figures
  • Translation-based nucleic acid alignment
  • ABI trace viewing, editing and printing

Let's see how to do a sequence alignment using bioedit software....

First thing you have to do is to get the sequences of your interest, it can be whole genome sequence or particular gene.This can be retrieved from NCBI (National Center for Biotechnology Information) .

For Example let's search for Haemagluttin gene (HA) of H1N1

You can save the selected gene sequence in FASTA format, which can be used in Bioedit software.

You can import multiple sequence files into one window
File > New Alignment. File> Import> Sequence alignment file> choose text file that save the FASTA sequences.

Once all the sequence have been selected you can run Clustal W from the Accessory application menu

choose all sequence> Accessory Application> ClastalW Multiple alignment> Run ClastalW> OK> Alignment> Find Conserved Regions> Start

The result will show how many conserved regions found and details of each region. Then choose one region as the template


Then use the chosen one region as template for real-time PCR primer and probe design. Before the region is used as template, checking specificity of this region by alignment of this region is required by using “Nucleotide BLAST”


Conserved regions are important because these regions will have least mutations, so these are ideal region for designing primers and probe for qPCR.

Primers can be Picked up from the conserved region using one of the many softtwares, to mention few

  • Primer 3
  • Primer Premier
  • NCBI
  • Oligo Designer, etc
References and Links:

Download BioEdit
BioEdit Help Files

Tuesday, September 3, 2013

Top 10 Blockbuster Drug Molecules!!!!

These molecules made the companies billion dollar worth...

Rank: 1

Trade Name: Lipitor 

INN: Atrovastatin

Company: Pfizer 

Technology: Chiral Chemistry 

Therapeutic Use: Cholesterol Lowering 

Sales : $125 Billion

Patent Expired : Novemeber 30, 2011

Rank 2

Trade Name: Plavix 

INN: Clopidogrel

Company: Sanofi/ Bristol 

Technology: Small Molecule Chemistry

Theraputic use: Anticlotting

Sales: $19.5 Billion, Patent Expiry: 2012

Rank 3

Trade Name: Advair

Company: Glaxo Smith Kine

Technology: Small Molecule Chemistry

Therapeutic Use: Asthma

Sales: $9.0 Billion

Patent Expiry: 2013 (European)

Rank 4

Trade Name:  Remicade

INN: Infliximab

Company: Merck,J&J

Technology: Monoclonal Antibody

Therapeutic Use: Arthritis

 Sales: $7.67 Billion, Patent Expiry: 2015

Rank: 5

Trade Name: Enbrel

Company: Pfizer (Wyeth)

Technology: Recombinant Gene Product

Therapeutic Use: Arthritis 

Sales: $7.1 Billion

Patent Expiry: 2012

Rank: 6

Trade Name: Humira

Company: Abbott

Technology: Monoclonal Antibody

Therapeutic Use: Arthritis

 Sales: $6.8 Billion

Patent Expiry: 2016

Rank 7

Trade Name: Avastin

Company: Roche

Technology: Monoclonal Antibody

Therapeutic Use: Cancer

Sales: $6.7 Billion

Rank 8

Trade Name: Rituxan

Company: Roche

Technology: Monoclonal Antibody

Therapeutic Use: Cancer

Sales: $6.1 Billion 

Rank 9

Trade Name: Diovan

Company: Novartis

Technology: Small Molecule Chemistry

Therapeutic Use: Hypertension

Sales: $ 6.0 Billion


Trade Name: Crestor

Company: Astrazeneca

Technology: Small Molecule Chemistry

Therapeutic Use: Cholesterol Lowering

Sales: $5.8 Billion