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Pierce™ Coomassie (Bradford) Protein Assay Kit Product

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Tissue - mouse brain

Products Thermo Fisher Scientific Pierce™ Coomassie (Bradford) Protein Assay Kit

Aurum™ Total RNA Fatty and Fibrous Tissue Kit Product

Get tips on using Aurum™ Total RNA Fatty and Fibrous Tissue Kit to perform RNA isolation / purification Tissue - Human Uterus

Products Bio-Rad Laboratories Aurum™ Total RNA Fatty and Fibrous Tissue Kit

Aurum™ Total RNA Fatty and Fibrous Tissue Kit Product

Get tips on using Aurum™ Total RNA Fatty and Fibrous Tissue Kit to perform RNA isolation / purification Tissue - Human Adipose

Products Bio-Rad Laboratories Aurum™ Total RNA Fatty and Fibrous Tissue Kit

cDNA synthesis Tissue Experiment

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

RNA cDNA synthesis Tissue

Protein isolation Mammalian cells - Mouse_Brown fat Experiment

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.

Proteins Protein isolation Mammalian cells Mouse_Brown fat

TRIzol Reagent Product

Get tips on using TRIzol Reagent to perform RNA isolation / purification Tissue - mouse lung tissue