Skip to main content

Protein Identification

There are several techniques which are used for the identification of a protein structure such as optical rotatory dispersion, X-ray crystallography, spectroscopy and nuclear magnetic resonance, these techniques are powerful in determining the structural features of protein but they require many hours of highly skilled and technically dedicated work environments. There are several computational techniques which allow for the biological discovery based on the protein sequences only and/or on their comparison to protein families. 

There are three main methods to identify protein and its structural features. 

1) Protein Identity Based on Composition: 

This involves the methods and online packages such as AA Compident, AA Compsim and Propsearch.. (AA stands for Amino Acid). 
AA Compident: It uses the amino acid composition of the unknown protein to identify known proteins of the same composition. 
AA Compsim: Instead of using the experimentally derived amino acid composition, it uses the sequence of a SWISS-PROT protein. A theoretical pI and molecular weight are computed. 
PropSearch: this method is robust and various physical properties are used in performing the analysis. 

2) Physical properties based on sequence:

There are 4 methods to identify the physical properties of a protein based on sequence. PeptideMass, TGREASE, SAPS and Compute pI/MQ and ProtParam.
PeptideMass: It is used for the peptide mapping experiments and it determines the cleavage products of a protein after treatment with protease or other chemical reagent. 
TGREASE: calculates the hydrophobicity of a protein along its length. 
SAPS: It provides the extensive statistical information for any given query sequence. It stands for Statistical Analysis of Protein Sequences. 
Computer pI/MW: It calculates the isoelectric point and molecular weight of an input sequence. 
ProtParam: It calculates the molecular weight, isoelectric point, overall amino acid composition, a theoretical extinction coefficient and grand average hydrophobicity.

3) Protein Structure Prediction: 

For the prediction of secondary structures of a given protein, several online servers like JPRED, PSIPRED are used. 

Using servers like Modeler, primer and Swiss prot, the homology modelling for protein sequences showing similarity to other sequences are predicted. 

I-Tasser and Rossetta are the Ab-initio structure prediction servers which are used for the protein sequences that hardly share any similarity to known protein structures. 
Labels:

Comments

Post a Comment

Popular posts from this blog

Statistics in Flow Cytometry Data and "MFI" values

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keep...
1 comment
....CONTINUE READING

Nanodrop

Thermofisher, Nanodrop 1000    Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm).  Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument.  It works on the principle of Spectrophotometer or Sample Retention System....  Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of colum...
Post a Comment
....CONTINUE READING

Protocol: Sample preparation for Cell Cycle Analysis by Flow Cytometry

Protocol:  Preparation of Samples for Cell Cycle Analysis by Flow Cytometry: 1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension.  2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells.  ( cells can kept at 4 ℃ for a week).  3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet.  4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette.  5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet.  6) if required, step 5 can be repeated for one more time.  7) Add 1mL of Propidium Iodide (PI...
Post a Comment
....CONTINUE READING