Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
[Abstract] The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different ...
Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
[Abstract] Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary ...
Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast
Authors: Juan Flores and
Nan Gao,
date: 10/20/2016,
view: 6527,
Q&A: 0
[Abstract] This protocol describes the procedure of visualizing secreted Wnt5 protein in serum free media via western blotting. This procedure can also be used to visualize other secreted proteins larger than 10,000 daltons. The work presented in this paper visualizes Wnt5 secreted by mouse embryonic fibroblast (MEF), but can be adapted to other cell lines ...
Detection of HBV C Protein Phosphorylation in the Cell
Authors: Jaesung Jung and
Kyongmin Kim,
date: 08/05/2015,
view: 6269,
Q&A: 0
[Abstract] Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine ...
Immunoprecipitation of Proteins in Caenorhabditis elegans
[Abstract] Immunoprecipitation (IP) is a biochemical technique to precipitate a protein out of solution using an antigen that can specifically bind to that protein. IP can be performed to isolate and concentrate one particular protein from a sample of thousands of different proteins. IP is also readily performed to pull down interacting proteins of complexes ...
Lectin Binding Analysis of Streptococcus mutans Glycoproteins
[Abstract] Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various ...
Enzymatic Reactions and Detection of C3 Cleavage Fragments
Authors: Claudia Kemper and
Martin Kolev,
date: 08/20/2014,
view: 9043,
Q&A: 0
[Abstract] The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 ...
Preparation of Parasite Protein Extracts and Western Blot Analysis
Authors: Arlett Heiber and
Tobias Spielmann,
date: 06/05/2014,
view: 20515,
Q&A: 0
[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available ...