ELISA Rat

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Renal tissue

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - Rat_Circumvallate papillae

Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - Rat_Liver

Get tips on using LC3A/B (D3U4C) XP® Rabbit mAb to perform Autophagy assay cell type - RAW 264.7

Get tips on using Anti-LC3B antibody produced in rabbit to perform Autophagy assay cell type - Proximal tubular cells (rPT)

Get tips on using LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082 to perform Autophagy assay cell type - RAW 264.7

Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?

A gross majority of classical apoptotic attributes can be quantitatively examined by flow cytometry, the preferred platform for rapid assessment of multiple cellular attributes at a single-cell level. However, sample preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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.