Protocols in Current Issue
    Capillary Nano-immunoassay for Quantification of Proteins from CD138-purified Myeloma Cells
    Authors:  Irena Misiewicz-Krzeminska, Isabel Isidro and Norma C. Gutiérrez, date: 06/20/2019, view: 1238, Q&A: 0
    [Abstract] Protein analysis in bone marrow samples from patients with multiple myeloma (MM) has been limited by the low concentration of proteins obtained after CD138+ cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each CD138+ purified MM sample in an ...
    Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
    Authors:  Rubén Martínez-Barricarte, Xiao-Fei Kong and Jean-Laurent Casanova, date: 06/05/2019, view: 521, Q&A: 0
    [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
    Authors:  Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 2551, Q&A: 0
    [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: 4662, 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: 5006, 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
    Authors:  Kevin K. Chan, Ashwin Seetharaman, Guillermo Selman and Peter John Roy, date: 04/05/2015, view: 11040, Q&A: 1
    [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
    Authors:  Alejandro Avilés-Reyes, José A. Lemos and Jacqueline Abranches, date: 04/05/2015, view: 5569, Q&A: 0
    [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 ...
    Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
    Authors:  Brigitte ME Hayes and Nicole L van der Weerden, date: 09/20/2014, view: 5953, Q&A: 0
    [Abstract] It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans ( ...
    Enzymatic Reactions and Detection of C3 Cleavage Fragments
    Authors:  Claudia Kemper and Martin Kolev, date: 08/20/2014, view: 7022, 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: 16406, 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 ...

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