Cellular elements are organized into membrane-bound and non-membrane bound structures called organelles as shown in the figure below. Every organelle has a distinct cellular structure with a specified biological function. Each function is determined by the enzymes or proteins the organelle contains. Each organelle performs explicit functions such as providing the shape and support for the cell, energy production, processing, transportation/storage of biomolecules, and DNA maintenance—to name a few.
Cell and organelle marker antibodies are utilized mostly for the determination of the localization of novel proteins within complex structures, and aid in discerning the proximity of two proteins within the same subcellular assembly. They assist in analyzing alterations in protein localization and transport to provide insights on protein function and disease mechanisms. Cell and organelle marker antibodies also help monitor and provide information on the biochemical purity of organelle fractionation.
The following sections briefly summarize the characteristics and function of the major cellular structures—the cell membrane, nucleus, and cytoplasm. Each section provides data examples using well-characterized marker antibodies.
The cell membrane is a selectively permeable bilayer of phospholipid molecules that separates the intracellular components from the extracellular environment. Proteins specific to the membrane provide structural support, aid in cell signaling, anchor the cytoskeleton, and control the passage of materials in and out of the cells.
Proteins specific to the plasma membrane and cell junctions, such as ZO-1, ZO-3, and E-cadherin, serve as markers that help study and clarify the role of novel proteins which may act as receptors, transport channels, and/or enzymes. The antibodies represented in the figure below have been verified for target specificity using independent antibody validation.
Figure 2. Immunofluorescence analysis of cell membrane proteins in CaCo-2 and MCF7 cells. ZO-1 (panel a, d), ZO-3 (panel b, e), and E-cadherin (panel c, f) was performed using ZO-1 Polyclonal Antibody (Cat. No. PA5-28869), ZO-3 Recombinant Rabbit Monoclonal Antibody (24H3L15) (Cat. No. 701825) and E-cadherin Monoclonal Antibody (EP700Y) (Cat. No. MA5-14458) on Caco-2 (ZO-1, ZO-3) and MCF7 (E-cadherin) cells. Each was labeled with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034). Panels d, e, and f are composite images that clearly demonstrate membrane and junctional localization of ZO-1, ZO-3, and E-cadherin proteins, respectively.
The nucleus is a bilipid membrane-bound organelle encompassing the genetic material of the cell. Components of the nucleus include:
Antibodies targeting distinct proteins in the nucleus, like histones H3 and H4, LSD1, fibrillarin, lamin B1, and CENPA, serve as marker proteins that facilitate the study of morphology and dynamics of the nucleus and its constituents. Histone and histone-associated proteins involved in DNA compaction, chromatin regulation, and gene expression—such as H3, H4, and LSD1—function as markers for the chromatin and nucleus, while the histone H3 variant CENPA specifically serves as a marker for centromeres. Lamins B1, A, and C function as structural support to the nucleus and are involved in the regulation of transcription. All of these are good marker choices for studying the nuclear membrane. Fibrillarin, which is involved in the regulation of RNA transcription and processing, is best utilized as a nucleoli marker.
Figure 3. Immunofluorescence analysis of nuclear proteins in HeLa and NIH/3T3 cells. Immunofluorescence analysis was performed on fixed and permeabilized HeLa (panel a, b, c) and NIH/3T3 (panel d) cells for detection of endogenous LSD1 (panel a), lamin B1 (panel b), fibrillarin (panel c), and CENPA (panel d) using LSD1 Recombinant Rabbit Monoclonal Antibody (3H8L63) (Cat. No. 703761), Lamin B1 Recombinant Rabbit Monoclonal Antibody (10H34L18) (Cat. No. 702972), Fibrillarin Monoclonal Antibody (38F3) (Cat. No. 480009), and CENPA Monoclonal Antibody (H.577.2) (Cat. No. MA5-14829), respectively. Panel a demonstrates nuclear localization of LSD1, panel b shows nuclear membrane localization of lamin B1, panel c represents fibrillarin located in the nucleolus, and panel d depicts centromere localization of CENPA protein. The images were captured at 60X magnification.
Each of the antibodies used in the above immunofluorescence experiment were validated for use in that application. Also, antibody specificity was demonstrated by siRNA-mediated knockdown of the target proteins. The western blots below are the data from that verification study.
Figure 4. Western blot analysis of siRNA-mediated knockdown of nuclear proteins in MCF7, HeLa, and NIH/3T3 cells. Knockdown of LSD1 (panel a), lamin B1 (panel b), fibrillarin (panel c), and CENPA (panel D) was achieved by transfecting cells with respective protein-specific siRNAs. Western blot analysis was performed on MCF7 (LSD1), HeLa (lamin B1, fibrillarin), and NIH/3T3 (CENPA) cell extracts from the knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2), and untransfected cells (Lane 1). The blots were probed using LSD1 Recombinant Rabbit Monoclonal Antibody (3H8L63) (Cat. No. 703761), Lamin B1 Recombinant Rabbit Monoclonal Antibody (10H34L18) (Cat. No. 702972), Fibrillarin Monoclonal Antibody (38F3) (Cat. No. 480009), and CENPA Monoclonal Antibody (H.577.2) (Cat. No. MA5-14829), respectively.
The cytoplasm consists of organelles suspended in the cytosol where most of the cellular activities occur. The organelles of the cytoplasm can be membrane- or non-membrane-bound and have a definite structure and specific cellular function. The organelles of the cytoplasm and their function are in the table below, along with their target marker antibodies.
Cytoplasmic organelle antibody targets:
|Mitochondria||Enables cellular respiration and regulates cellular metabolism||HK1, HSP60, Cytochrome C|
|Endoplasmic reticulum||Responsible for protein synthesis, folding, and secretory protein transport||Calnexin, Calreticulin, PDI|
|Golgi apparatus||Sorting station for vesicular trafficking and helps in packaging and modification of proteins and lipids||GM130, TGN46,|
Golgi protein 58K
|Autophagosome||Assists in the breakdown of biomolecules by process of fusion with lysosomes||ATG5, ATG12, LC3B|
|Lysosomes||Involved in the breakdown of all types of biomolecules||LAMP1, LAMP2, LIMP2|
|Peroxisome||Facilitates enzymatic oxidative reaction and catalyzes the breakdown of hydrogen peroxide||Catalase|
|Ribosome||Primary location for protein synthesis||RPS3, RPS6, RPL7A|
|Proteasome||Degrades damaged or unwanted proteins by proteolysis||PSMA1, PSMD1, PSMD7|
Organelle marker antibodies targeting diverse cytoplasmic constituents help clarify the organization of subcellular structures, function, and the role of novel proteins in different cellular processes. Below are some examples of antibody targets in various organelles and the antibodies that are specific and verified to detect them.
Catalase is a key peroxisomal marker protein and functions to protect the cells from the toxic effects of hydrogen peroxide. Golgi protein, GM130, is involved in multiple tasks including ER-Golgi transport, structural maintenance, and stacking of Golgi cisternae. The specificity of the catalase and GM130 antibodies was verified in a biological setting utilizing the cell’s natural machinery to effectively knock down the target gene expression as shown in the figure below.
Figure 5. Immunofluorescence analysis of siRNA-mediated knockdown of catalase and GM130 in HepG2 and HeLa cells. Immunofluorescence analysis was performed on untransfected cells (panel a, d), non-specific scrambled siRNA (panels b, e), and transfected cells with target-specific siRNA (panel c, f). Catalase Recombinant Rabbit Monoclonal Antibody (10H47L17) (Cat. No. 702955) and GM130 Recombinant Rabbit Monoclonal Antibody (10H5L5) (Cat. No. 703794) were used, respectively. Loss of signal was observed upon siRNA-mediated knockdown (panel c, f) confirming the specificity of the antibodies to catalase and GM130. The images were captured at 60X magnification.
Hexokinase 1, an enzyme kinase involved in the first step of the glycolysis, and LIMP2, a lysosomal receptor, act as markers for the outer mitochondrial membrane and lysosomes, respectively. The specificity of the hexokinase 1 (HK1) and LIMP2 antibodies was verified by co-localization experiments using organelle-specific stains. The data is shown below.
Figure 6. Immunofluorescence analysis of hexokinase 1 and LIMP2. Immunofluorescence analysis of hexokinase 1 (panel a-c) and LIMP2 (panel d-f) was performed using HK1 Recombinant Polyclonal Antibody (Cat. No. 711777, 1:100 dilution) and LIMP2 Recombinant Polyclonal Antibody (Cat. No. 712072, 1:100 dilution). Panel c and panel f show colocalization of hexokinase 1 with a mitochondrial marker, MitoTracker Red CMXRos (Cat. No. M-7512) and LIMP2 with lysosome marker, LysoTracker Red DND-99 (Cat. No. L-7528), demonstrating antibody specificity.
RPS6 is a part of the 40S subunit of the ribosome. It is a major substrate of protein kinases in the ribosome and is used as one of the markers for ribosomes. Cytochrome C is a key subunit involved in the mitochondrial electron transport chain and acts as a marker for the inner mitochondrial membrane.
Specificity of both targets is represented in the figures below.
Figure 7. Immunofluorescence analysis of cytochrome C and S6 ribosomal protein in HeLa and 3T3 cells. Immunofluorescence analysis of cytochrome C (panel a-c) and S6 ribosomal protein (panel d-f) were performed using HeLa and 3T3 cells, respectively. Cells were stained with a Cytochrome C Monoclonal Antibody (6H2.B4) (Cat. No. 33-8200) or S6 Recombinant Polyclonal Antibody (9HCLC) (Cat. No. 710405) and nuclei (blue) were stained with Hoechst 33342 Solution (20 mM) (Cat. No. 62249). Panel c represents cytochrome C located in the inner mitochondrial space and panel f demonstrates ribosomal localization of RBS6 protein. Images were taken at 20X magnification.
Figure 8. IP-MS enrichment of cytochrome C (LFQ intensity). Cytochrome C was enriched 489-fold from MCF7 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Cat. No. 90409) and Cytochrome C Monoclonal Antibody (6H2.B4) (Cat. No. 33-8200).
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