Protein Expression Eukaryotic cells W. anomalus

DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.

The most widely used method for protein quantification is by spectrophotometry. The concentration of the protein in the samples is measured at an absorbance of 280 nm. The absorbance of the sample protein is then plotted against a standard curve. This method allows for total protein quantification in a sample (cell and tissue extracts). Before analysing the concentration of protein in the sample, it is important to choose the right test method.  For high protein concentration samples (above 5 - 160 mg/ml) the best method is to use the Biuret test. For low concentrations samples (between 1 - 2000µg/ml) the best methods are Lowry assay, BCA assay, Bradford assay and coomassie blue (for exact sensitivity of the test kits you use, refer to manufacturer's protocol). If the samples contain detergents like Triton X-100 then BCA assay is the best choice. For samples that have proteins larger than 3 KDa in size Bradford assay is the best choice. Each method has advantages and disadvantages, plan your analysis considering your sample characteristics.

The most widely used method for protein quantification is by spectrophotometry. The concentration of the protein in the samples is measured at an absorbance of 280 nm. The absorbance of the sample protein is then plotted against a standard curve. This method allows for total protein quantification in a sample (cell and tissue extracts). Before analysing the concentration of protein in the sample, it is important to choose the right test method.  For high protein concentration samples (above 5 - 160 mg/ml) the best method is to use the Biuret test. For low concentrations samples (between 1 - 2000µg/ml) the best methods are Lowry assay, BCA assay, Bradford assay and coomassie blue (for exact sensitivity of the test kits you use, refer to manufacturer's protocol). If the samples contain detergents like Triton X-100 then BCA assay is the best choice. For samples that have proteins larger than 3 KDa in size Bradford assay is the best choice. Each method has advantages and disadvantages, plan your analysis considering your sample characteristics.

Get tips on using Mouse Gene Expression v2 4x44K Microarray Kit to perform Microarray Gene expression arrays - Mouse liver tissue Cyanine-3-CTP

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid-mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time-consuming, but provide a more permanent expression of RNAi in the cells and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines.

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with the desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid-mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time-consuming, but provide a more permanent expression of RNAi in the cells and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines.

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid-mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time-consuming, but provide a more permanent expression of RNAi in the cells and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines.

Get tips on using Qubit™ Protein Assay Kit to perform Protein quantification Mammalian cells - HeLa

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.