免疫印迹法(WB ) 实验方案 | 免疫检测 | 蛋白质 | 生物化学

Monitoring Endocytosis of Integral Membrane Proteins Using Western Blot-Based Detection of Biotinylated Antibody Uptake

利用基于 Western blot 的生物素化抗体内吞检测法监测膜整合蛋白的内吞过程

AG Alexandra Graninger

PS Prasanna Satpute-Krishnan

2467 Views

Nov 20, 2025

The antibody-uptake assay is a commonly used technique to monitor endocytosis of integral membrane proteins including transmembrane and glycosylphosphatidylinositol-anchored proteins (GPI-APs). The antibody-uptake assay typically involves incubating live cells with fluorophore-conjugated antibodies directed against the extracellular domain of the integral membrane protein of interest. Antibody uptake is then detected by flow cytometry or confocal microscopy. However, these detection modalities may be inaccessible to some labs or require extensive training to operate. Thus, we developed an easy and novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot-based approach to the antibody-uptake assay that exploits the strong affinity between biotin and streptavidin. Instead of incubating cells with fluorophore-conjugated antibodies to monitor antibody uptake, our assay involves incubating cells with biotinylated antibodies, processing the cell lysates for western blot, and probing the membrane with detectably conjugated streptavidin. From preparation to quantification, this protocol requires less hands-on time than other approaches and is amenable to small-scale drug or siRNA screens. Here, we demonstrate the utility of our approach using the well-characterized misfolded GPI-AP, YFP-tagged C179A mutant of prion protein (YFP-PrP*), as our model substrate. YFP-PrP* constitutively traffics to the plasma membrane (PM), where it binds to anti-GFP antibody, and immediately undergoes endocytosis to lysosomes. To validate our protocol, we present measurements of antibody uptake under conditions known to enhance or inhibit YFP-PrP*’s traffic to the PM. Using this assay, we present new evidence that, under certain conditions, YFP-PrP* is able to undergo degradation via a pathway that does not involve exposure on the cell surface.

Use of a High-Affinity Ubiquitin-Binding Domain to Detect and Purify Ubiquitinated Substrates and Their Interacting Proteins

利用高亲和力泛素结合结构域检测和纯化泛素化底物及其互作蛋白

NS Nitu Saha

MZ Mengwen Zhang

MH Mark Hochstrasser

0 Q&A

4007 Views

Sep 5, 2025

OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism Orientia tsutsugamushi. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography–tandem mass spectrometry (LC–MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology.

Far-western Blotting Detection of the Binding of Insulin Receptor Substrate to the Insulin Receptor

Far-western blotting 检测胰岛素受体底物与胰岛素受体的结合

JP Jinghua Peng

BR Balamurugan Ramatchandirin

AP Alexia Pearah

LH Ling He

0 Q&A

2596 Views

Feb 20, 2023

Far-western blotting, derived from the western blot, has been used to detect interactions between proteins in vitro, such as receptor–ligand interactions. The insulin signaling pathway plays a critical role in the regulation of both metabolism and cell growth. The binding of the insulin receptor substrate (IRS) to the insulin receptor is essential for the propagation of downstream signaling after the activation of the insulin receptor by insulin. Here, we describe a step-by-step far-western blotting protocol for determining the binding of IRS to the insulin receptor.

Capillary Nano-immunoassay for Quantification of Proteins from CD138-purified Myeloma Cells

毛细管纳米免疫实验定量分析经CD138筛选的骨髓瘤细胞蛋白

IM Irena Misiewicz-Krzeminska

II Isabel Isidro

NG Norma C. Gutiérrez

0 Q&A

6998 Views

Jun 20, 2019

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 automated manner. Up to now, the knowledge of protein level in those cells was limited because a relatively small quantity of sample is available after the diagnostic procedure. Moreover, the sample often is required for nucleic acids analysis. We have developed the procedure for obtaining proteins from bone marrow samples preserved in RLT+ buffer, and we have successfully applied this approach for the quantification of proteins in the setting of patients with MM. Proteins are extracted from RLT+ buffer, the content is quantified by total protein assay with WES machine and finally, the particular protein expression level is evaluated using specific antibodies by capillary nano-immunoassay with WES machine. The present protocol enables us to quantify many proteins from a limited amount of sample, without losing the opportunity to obtain nucleic acids at the same time. Proteins are quantified automatically in an assay with a low probability of human errors, which makes it a useful tool for biomarkers development.

Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor

SPPL2a抑制剂处理细胞后的CD74N端片段聚集的检测

RM Rubén Martínez-Barricarte

XK Xiao-Fei Kong

JC Jean-Laurent Casanova

0 Q&A

7063 Views

Jun 5, 2019

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 subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.

Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons

Neuro2A细胞和大鼠原代皮层神经元中突变亨廷顿蛋白的分泌

KT Katarina Trajkovic

HJ Hyunkyung Jeong

DK Dimitri Krainc

0 Q&A

8674 Views

Jan 5, 2018

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 cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration.

Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast

检测小鼠胚胎成纤维细胞调节培养基中的Wnt5

JF Juan Flores

Nan Gao

0 Q&A

9095 Views

Oct 20, 2016

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 including those transiently transfected by plasmids.

Detection of HBV C Protein Phosphorylation in the Cell

细胞中的HBV C蛋白磷酸化位点检测

JJ Jaesung Jung

Kyongmin Kim

0 Q&A

8715 Views

Aug 5, 2015

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 whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with ³²P-orthophosphate for 14 h, and then lysed. The ³²Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the ³²Pi-labeled immunoprecipitated C proteins were detected by autoradiography.

Immunoprecipitation of Proteins in Caenorhabditis elegans

免疫沉淀法检测秀丽隐杆线虫蛋白质

KC Kevin K. Chan

AS Ashwin Seetharaman

GS Guillermo Selman

PR Peter John Roy

1 Q&A

18563 Views

Apr 5, 2015

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 out of solution. This protocol outlines the methods used to IP proteins in whole worm lysates and their preparation for detection on Western blots using denaturing conditions.

Lectin Binding Analysis of Streptococcus mutans Glycoproteins

变异链球菌糖蛋白的凝集素结合分析

Alejandro Avilés-Reyes

José A. Lemos

Jacqueline Abranches

0 Q&A

9674 Views

Apr 5, 2015

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 techniques, this is often limited by the specific characteristics of individual proteins such as type and level of glycosylation. Lectins are sugar-binding proteins that recognize specific glycoconjugates in a manner similar to antigen-antibody interactions. Here, we describe a simple method for the detection of glycoproteins using lectin-based Western blot analysis, which can be applied to different organisms and coupled with various other strategies for complementary analysis.