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Friday, June 28, 2013

Protein Characterization : Methods & Techniques

Protein Characterization: Protein characterization is primarily focused on the analysis of recombinant proteins for identifying and generating peptide maps of proteins which will aid in the purification process. Protein characterization can be done using the following methods:
  • Amino Acid Composition Analysis (AAA)
Determination of amino acid composition of a protein. Used for confirmation of primary structure.
  • N- and C-terminal Sequencing
N and C terminal protein sequencing

Determination of amino acid sequence from the N- and C-terminus of protein using Edman sequencing and/or MALDI-ISD. Used as a confirmation of primary structure or de novo sequencing.
  • 1D/2D SDS-PAGE
Detection of mono- and multimeric forms of protein and impurities.

SDS PAGE analysis

  • (Capillary) Isoelectric Focusing (IEF + cIEF)
Determination of the isoelectric point (pl) of a protein and assessment of isoform distribution and impurity profiling.
  • Western Blot
Identification of proteins by their immunologic reaction with antibodies of known specificity (from 1D and 2D PAGE gels).
Separation of protein based on size. Determination of degree of aggregation.
  • RP and IEX-HPLC
Identity, content and impurity determination. Detection of oxidation and deamidation products.
  • Peptide Mapping (by LC-MS) and MS/MS (de novo) Protein Sequencing
Used to confirm identity/primary structure. Enzymatic digestion of protein followed by mass spectrometry.
  • Monosaccharide Composition
Quantification of neutral amino sugars: glucosamine, galactosamine, mannose, galactose and fucose.

Carbohydrate Linkage Analysis Determination of monosaccharide linkage variants as part of a glycan structure analysis.
  • Sialic Acid Determination
Quantitation of acidic sugars NANA (Neu5Ac) and NGNA (Neu5Gc).
  • Oligosaccharide Profiling
N-linked and O-linked oligosaccharide profiling including analysis of sialylated and desialylated carbohydrate structures by NP-HPLC (HILIC), HPAEC-PAD and mass spectrometry.
Detection and quantitation of residual proteins including BSA, Protein A and host cell proteins. Proprietary HCP assays for CHO and E. coli; also useful in bioassays and protein characterization.

ELISA Protein detection

  • Extinction Coefficient
Determination of extinction coefficient and content of protein by UV and Amino Acid Analysis.
  • Determination of Protein Content
Routine determination by UV or other methods.
  • Glycosylation/Sulfation/Phosphorylation Site Identification
Mass spectrometry analysis to determine the site of post-translational modifications.
  • Disulfide Bridge Analysis
Cystine linkage sites are identified (de novo assessment and/or confirmation).
  • Oxidation and Deamidation Analysis
Mass spectrometry is used for detailed characterization of extent and site of modification.
  • Presentation-Specific Testing
Physicochemical testing as required for the product type (e.g., osmolality, sub-visible particulates, moisture content).
  • Capillary (Zone or SDS) Electrophoresis
Used for the characterization and separation of protein mixtures based on mass or charge.

Thursday, June 27, 2013

Melting Curve Analysis on Real Time PCR

Melting Curve analysis in qPCR is a method of analyzing the dissociation of double stranded DNA during the  heating cycles. The double stranded DNA starts separating out from each other when the temperature is raised, which in-turn causes the change in the intensity of absorbance. melting curve shouldn't be confused with the melting point which denoted by Tm.

Melt curve analysis method can be used to check for any primer dimer formation and other anomalies in the qPCR assay developed.

Melting Temperatures varies from DNA's of different species based on the GC content, more GC content more the temperature required to melt. The reason for this is due to the presence of 3 hydrogen bonds in the GC Pairing as compared to two hydrogen bonds in the AT pairing. So more energy is to break the GC pair.

The temperature dependent dissociation of DNA strands can be measured by using intercalating flourophores like SYBR green, EVA Green or other fluorescent labelled probes. SYBR green binds to the double stranded DNA emits fluorescence, when the temperature is increased DNA denatures / dissociates and becomes single stranded marked by reduction in the fluorescence.

DNA melt curve real time PCR

The graph of the negative first derivative of the melting-curve may make it easier to pin-point the temperature of dissociation (defined as 50% dissociation), by virtue of the peaks thus formed.

One of the other advantage of using the melt curve analysis method is that to find the Single nucleotide Polymerphisms (SNPs).

Monday, June 24, 2013

MIQE Guidelines for representing and analysing qPCR data

MIQE guidelines are a set of guidelines for the analysis and representation of qPCR or Real Time PCRdata, inaccurate data and statistical analysis can be misleading and one need to follow the MIQE guidelines for representing and analysing qPCR data

Standard terms to be used for Real Time PCR data publications:

  • qPCR for quantitative PCR and RT – PCR for reverse transcription PCR. The term RT-PCR should not be used to represent real time PCR.

  • genes used for normalization should be referred to as reference genes not house keeping genes.

  • TaqMan probes should be referred to as hydrolysis probe.

  • The term FRET probe (fluorescence resonance energy transfer probe) refers to a generic mechanism in which emission/quenching relies on the interaction between the electron-excitation states of 2 fluorescent dye molecules. LightCycler-type probes should be referred to as dual hybridization probes.

  • Quantification is the correct term to use not quantitation.

  • According to RDML (Real-Time PCR Data Markup Language) data standard quantification cycle (Cq) is the correct term to use, threshold Cycle Ct, Crossing point (Cp) and take-off point(TOP) even though all these are coined by manufactures of PCR machines for differentiation.

Analytical sensitivity refers to the minimum number of copies in a sample that can be measured accurately with an assay, whereas clinical sensitivity is the percentage of individuals with a given disorder whom the assay identifies as positive for that condition. Typically, sensitivity is expressed as the limit of detection (LOD), which is the concentration that can be detected with reasonable certainty with a given analytical procedure. The most sensitive LOD theoretically possible is 3 copies per PCR.

Analytical specificity refers to the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample. Diagnostic specificity is the percentage of individuals without a given condition whom the assay identifies as negative for that condition.

Accuracy refers to the difference between experimentally measured and actual concentrations, presented as fold changes or copy number estimates.

Repeatability (short-term precision or intraassay variance) refers to the precision and robustness of the assay with the same samples repeatedly analyzed in the same assay. It may be expressed as the SD for the Cq variance. Alternatively, the SD or the CV for copy number or concentration variance may be used. CVs should not be used with Cqs.

Reproducibility (long-term precision or interassay variance) refers to the variation in results between runs or between different laboratories and is typically expressed as the SD or CV of copy numbers or concentrations. Cq values generated from different runs are subject to inherent interrun variation hence, reporting interrun Cq variation is not appropriate.


MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Special Report

Sunday, June 23, 2013

MIQE Guidelines : RDML Grahical Schema for qPCR data Publications

MIQE refers to Minimum Informaion for Quantitative Real-Time PCR Experiments. The Image below depicts the graphical schema format for data publications of Real Time PCR experiments.The RDML file format was created to encourage the exchange, publication, revision and re-analysis of raw qPCR data.

RDML stands for Real Time PCR Data Markup Language

real time PCR data reporting guidelines

Real Time PCR Data Markup Language (RDML)