Site Directed Mutagenesis (SDM) Human Point mutation BxPC-3

Get tips on using PI 3-kinase p85α Antibody (B-9) to perform Autophagy assay cell type - SH-SY5Y

Get tips on using 3 µm Chemotaxis Assays, 96-Well Format to perform Cell migration / Invasion cell type - Jurkat

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

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.

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

Get tips on using Oct-3/4 Antibody (C-10): sc-5279 to perform Western blotting Oct4

Get tips on using OxiSelect™ Comet Assay Kit (3-Well Slides) to perform DNA Damage Assay HeLa

Get tips on using OxiSelect™ Comet Assay Kit (3-Well Slides) to perform DNA Damage Assay MCF 10A

Get tips on using OxiSelect™ Comet Assay Kit (3-Well Slides) to perform DNA Damage Assay Hep G2