Antibodies to Ubiquitin-like Proteins - FocusOn 024

Introduction: Ubiquitin-like proteins fall into two classes: Ubiquitin-like modifiers (UBLs) and Ubiquitin-domain proteins (UDPs).

The first class, ubiquitin-like modifiers, function as modifiers in a manner analogous to that of Ubiquitin, which influences protein turnover in a cell by closely regulating the degradation of specific proteins (please see “Focus On Ubiquitin”). Examples of UBLs are SUMO, Rub1 (also called Nedd8), Apg8 and Apg12.

Proteins of the second class include parkin, RAD23 and DSK2, are designated ubiquitin-domain proteins. These proteins contain domains that are related to ubiquitin but are otherwise unrelated to each other. In contrast to UBLs, UDPs are not conjugated to other proteins.

Apg12 is synthesized in its mature form and seems to have one target, Apg5. Almost all Apg12 molecules are conjugated with Apg5. In yeast, autophagy, the delivery of cytoplasmic components to the lysosome/vacuole for degradation, requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg12-Apg5 and Apg16.

Apg8 exists in two forms, one is membrane bound through a phospholipid. Lipidation/ activation of Apg8 is mediated by Apg7 and transferred to Apg3 and finally forms a conjugate with phosphatidyl-ethanolamine (PE). Apg4 cleaves Apg8–PE, releasing Apg8 from membrane. Morphological studies show that Apg8 localizes on the membrane of intermediate structures of the autophagosome; this transient association seems to be essential for formation of the autophagosome.

Hub1 ("Homologous to Ubiquitin") may function as a modifier (see fig. 1) but its role is unclear because it lacks the double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. Recently cell polarity factors Sph1 and Hbt1 have been identified as in vivo targets of Hub1 conjugation. HUB1 has close homologs in other species including humans. The human homologs is called Ubl5.

Rub1 and the corresponding human homolog Nedd8 are activated by the E1 ubiquitin-activating enzyme UBA2, that forms isopeptide linkages between thio esters. Rub1 modification may regulate SCF function or localization.

Urm1 is a newly identified ubiquitin related modifier. Urm 1 is a 99-amino acid protein terminated with glycine-glycine. Target proteins are conjugated to Urm1 via its C-terminal glycine. Initially Urm1 forms a thioester with a novel E1-like protein, Uba4

ISG15 (Interferon Stimulating Gene-15) also known also as UCRP (ubiquitin cross-reactive protein) shows no amino acid sequence homology to cytokines and is synthesized as a precursor that is activated through processing by a thiol protease. ISG15 is secreted by monocytes and lymphocytes. ISG15 has been shown to modulate immune cell function. It possesses activities of cytokines and induces production of IFN-gamma. It enhances proliferation and functions of natural killer and LAK cells.

Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO (small ubiquitin-like modifier) regulates various cellular processes, such as nuclear transport, signal transduction, stress responses and cell cycle progression. But, in contrast to ubiquination, sumoylation does not tag proteins for degradation by the 26S proteasome, but rather seems to enhance stability or modulate their subcellular compartmentalization.

 Figure 1. Conjugation pathways for ubiquitin and UBLs. Most modifiers mature by proteolytic processing from inactive precursors (a; amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC) -like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin.

Fig.1: Conjugation pathways for ubiquitin and UBLs. Most modifiers mature by proteolytic processing from inactive precursors (a; amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC) -like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin.

Antibody Tools for the Detection of Ubiquitin-like proteins

Acris Antibodies offers a wide range of antibodies for the detection of proteins belonging to the Ubiquitin-like modifier family. These antibodies are available in various formats and can be used in immunological methods like Western blotting or ELISA.

Figure 2. Immunoblot of Hub1 fusion protein.</strong> Anti-Hub1 antibody generated by immunization with recombinant yeast Hub1 was tested by immunoblot against yeast lysates expressing the Hub1-GFP fusion protein and other UBL fusion proteins. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of Acris anti-GFP (code #R1091P) followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG (code # R1254HRP). Panel C shows specific reaction with Hub1 using a 1:500 dilution of Acris IgG fraction of Rabbit-anti-Hub1 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG (code # R1454HRP). All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4°C. Yeast lysate proteins were separated by SDS-PAGE using a 15% gel. This data indicates that anti-Hub1 is highly specific and does not cross react with other UBLs

 

Fig.2: Immunoblot of Hub1 fusion protein. Anti-Hub1 antibody generated by immunization with recombinant yeast Hub1 was tested by immunoblot against yeast lysates expressing the Hub1-GFP fusion protein and other UBL fusion proteins. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of Acris anti-GFP (code #R1091P) followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG (code # R1254HRP). Panel C shows specific reaction with Hub1 using a 1:500 dilution of Acris IgG fraction of Rabbit-anti-Hub1 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG (code # R1454HRP). All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4°C. Yeast lysate proteins were separated by SDS-PAGE using a 15% gel. This data indicates that anti-Hub1 is highly specific and does not cross react with other UBLs

Available Antibody Panel to UBLs

APG5L, APG8, APG12, Hub1, Rub1, URM1 (Urm1), ISG15, SUMO (Sumo), SUMO-1 (Sumo1), SUMO-2/3 (Sumo2/3), SUMO-3 (Sumo3) antibody.



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Primary Antibodies

Catalog No. Host Iso. Clone Pres. React. Applications  

APG5L / ATG5 (incl. pos. control) antibody

Monoclonal antibody ATG5-7C6 detects the endogenous Atg5 as well as the overexpressed 33 kDa-protein in Jurkat cells. 
Cells were fixed with 4% paraformaldehyd for 5 min and permeabilized with 0.05% Saponin. After 10 min aceton-treatment, Atg5 was detected with mab ATG5-7C6 at 0.5 µg/ml.  Images by courtesy of Hans-Uwe Simon, MD, PhD, University of Bern. Mouse IgG1 7C6 Purified Can, Hu, Ms, Rt F, ICC/IF, WB
0.1 mg / €430.00
  Acris Antibodies GmbH

APG5L / ATG5 (incl. pos. control) antibody

Whole lysate of serum starved SH-SY5Y cells was applied to SDS-PAGE and tranferred to a PVDF membrane. The Immunoblot was probed with the indicated monoclonal antibodies at 0.5 µg/ml for 1h at 15-22°C and developed with ECL (exposure time: 30 sec).
Lane M: Molecular Weight marker
Lane 1: AM20205PU-N (Clone 7C6)
Lane 2: AM20206PU-N (Clone 11C3) Mouse IgG1 11C3 Purified Can, Hu, Ms, Rt WB
0.1 mg / €430.00
  Acris Antibodies GmbH

APG5L / ATG5 (N-term) antibody

Kidney: Formalin-Fixed Paraffin-Embedded (FFPE) Rabbit IgG Aff - Purified Bov, Hu, Ms, Por, Pufferfish, Rt, Ze E, P, WB
50 µg / €470.00
  Acris Antibodies GmbH

APG8 / ATG8 antibody

Figure 1. Immunoblot of APG8 fusion protein. Anti-APG8 antibody generated by immunization with recombinant yeast APG8 was tested by immunoblot with other anti-UBL antibodies against E.coli lysates expressing the APG8-GFP fusion protein. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows specific reaction with APG8 using a 1:4,000 and 1:8,000 dilution of IgG fraction of Rabbit-anti-APG8 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG MX. All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4° C. E.coli lysate proteins were separated by SDS-PAGE using a 15% gel. Similar experiments (data not shown), where other UBL fusion proteins were separated and probed with this antibody showed no reactivity of anti-APG8 with other UBLs. This data indicates that anti-APG8 is highly specific and does not cross react with other UBLs. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov, www.lifesensors.com, personal communication. Rabbit Purified Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

ATG12 / APG12 (N-term) antibody

Formalin-fixed and paraffin-embedded human lung carcinoma tissue reacted with Autophagy APG12L Antibody Cat.-No AP32178PU-N (N-term), which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. Rabbit Ig Purified Hu P, WB
0.4 ml / €370.00
  Acris Antibodies GmbH

ATG12 / APG12 antibody

Figure 1. Immunoblot of APG12 fusion protein. Anti-APG12 antibody generated by immunization with recombinant yeast APG12
was tested by immunoblot against yeast lysates expressing the APG12-GFP fusion protein and other UBL fusion proteins. All UBLs
possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody
against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing
recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of anti-GFP followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG MX. Panel C shows specific
reaction with APG12 using a 1:2,000 dilution of IgG fraction of Rabbit-anti-APG12 (Yeast) followed by reaction with a
1:15,000 dilution of HRP Goat-a-Rabbit IgG MX. All primary antibodies were diluted in TTBS buffer supplemented
with 5% non-fat milk and incubated with the membranes overnight at 4° C. Yeast lysate proteins were separated by SDS-PAGE using a
15% gel. This data indicates that anti-APG12 is highly specific and does not cross react with other UBLs. A chemiluminescence
system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov,
Lifesensors Inc., personal communication. Rabbit Purified S. cerevisiae, Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

HUB1 antibody

Figure 1. Immunoblot of Hub1 fusion protein. Anti-Hub1 antibody generated by immunization with recombinant yeast Hub1 was tested by immunoblot against yeast lysates expressing the Hub1-GFP fusion protein and other UBL fusion proteins. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of anti-GFP followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG MX. Panel C shows specific reaction with Hub1 using a 1:500 dilution of IgG fraction of Rabbit-anti-Hub1 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG MX. All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4° C. Yeast lysate proteins were separated by SDS-PAGE using a 15% gel. This data indicates that anti-Hub1 is highly specific and does not cross react with other UBLs. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov, www.lifesensors.com, personal communication. Rabbit Purified Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

ISG15 / G1P2 (N-term) antibody

Formalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. Rabbit Ig Purified Hu, Ms P, WB
0.4 ml / €370.00
  Acris Antibodies GmbH

ISG15 / G1P2 (1-157) antibody

Western blot analysis: Cell lysates of HeLa (each 50 ug) were resolved by SDS-PAGE, transferred to PVDF membrane and probed with anti-human ISG15 (1:1000). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and an ECL detection system. Mouse IgG1 3E5 Purified Hu E, ICC/IF, WB
0.1 ml / €310.00
  Acris Antibodies GmbH

ISG15 / G1P2 (1-157) antibody

Western blot analysis: Cell lysates of HeLa (each 50 ug) were resolved by SDS-PAGE, transferred to PVDF membrane and probed with anti-human ISG15 (1:1000). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and an ECL detection system. Mouse IgG1 3E5 Purified Hu E, ICC/IF, WB
50 µl / €230.00
  Acris Antibodies GmbH

ISG15 / G1P2 (C-term) antibody

Formalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary antibody, followed by AEC staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. Rabbit Ig Purified Hu P
0.4 ml / €370.00
  Acris Antibodies GmbH

ISG15 / G1P2 antibody

Immunoblot of hISG15 fusion protein.
Anti-hISG15 antibody, generated by immunization
with recombinant human ISG15, was tested by immunoblot against a hISG15-GFP fusion protein produced in E.coli cell lysate soluble fraction.
Dilution of the antibody between 1:200 and 1:1,000
showed strong reactivity specifically with hISG15
and ISG15 coupled proteins. Free hISG15 is indicated by the arrowhead. In this blot the antibody was used at a 1:200 dilution incubated
overnight at 4° C in 5% non-fat dry milk in TTBS.
Detection occurred using a 1:2000 dilution of HRPlabeled
Donkey anti-Rabbit IgG for 1 hour at room temperature. A
chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M.Malakhov, personal Rabbit Purified Hu E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

RUB1 antibody

Figure. Immunoblot of Rub1 fusion protein. Anti-Rub1 antibody, generated by immunization with full-length recombinant yeast Rub1, was tested by immunoblot against a yeast cell lysate. A dilution of the antibody between 1:200 and 1:1,000 will show strong reactivity specifically with free Rub1 protein (indicated by arrow) and Rub1 conjugates. In this blot the antibody was used at a 1:500 dilution incubated overnight at 4°C in 5% non-fat dry milk in TTBS. Detection occurred using a 1:2000 dilution of HRP-labeled Donkey anti-Rabbit IgG for 1 hour at room temperature. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Rabbit Purified Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

SUMO1 antibody

Immunocytochemical detection of SUMO-1 on 4% PFA fixed HA-tagged SUMO-1 transfected 293T cells with AM26638AF-N. Mouse IgG1 5B12 Azide Free Hu, Ms, Rt ICC/IF, WB
0.1 mg / €340.00
  Acris Antibodies GmbH

SUMO1 (N-term) antibody

Immunocytochemical staining of HL-60 cells with Rabbit anti Human SUMO-1 (AP05599PU-N) Rabbit IgG Purified Hu, Ms, Rt ICC/IF, WB
0.1 mg / €330.00
  Acris Antibodies GmbH

SUMO1 antibody

Formalin-fixed and paraffin-embedded human brain tissue reacted with SUMO1 Antibody, which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. Rabbit Ig Aff - Purified Hu F, P, WB
0.4 ml / €370.00
  Acris Antibodies GmbH

SUMO1 antibody

Figure. Immunoblot of SUMO-GFP fusion proteins cleaved by insect cell protein extracts. R1198 SUMO antibody is generated by immunization with recombinant Yeast SUMO, was tested by Immunoblot against several constructs of SUMO-GFP fusion proteins after cleavage by proteases in insect cell protein extracts. These constructs contained various linkers between the SUMO and GFP portion of the fusion proteins. Each sample was run twice. The left Lanes each contain 2 µg E.coli expressed and purified SUMO-GFP fusion proteins after incubation with lysed cells (50 µg total protein) for 1 h. The right Lanes contain the same fusion proteins incubated with the lysate in the presence of 2% SDS. After probing with anti-GFP antibodies the membranes were stripped of antibody using SDS-DTT solution for 30 mn at 60°C and were then re-probed using the R1198 SUMO antibody at a 1/1000 dilution incubated overnight at 4°C in 5% non-fat dry milk in TTBS. Detection occurred using a 1/2000 dilution of HRP-labeled Donkey anti-Rabbit IgG for 1 h at RT. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Rabbit Purified Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

SUMO2 antibody

Immunoflourescence detection of SUMO2/3 on 4% PFA fixed SUMO2/3 transfected 293T cells with AM26639AF-N Mouse IgG2b 1E7 Azide Free Hu, Ms, Rt
0.1 mg / €350.00
  Acris Antibodies GmbH

SUMO2 (C-term) antibody

Formalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary antibody, followed by AEC staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. Rabbit Ig Purified Hu P, WB
0.4 ml / €370.00
  Acris Antibodies GmbH

SUMO2/3 antibody

Immunohistochemistry: Paraffin embedded sections of colorectal cancer tissue were incubated with anti-human SUMO2/3 antibody (1:50) for 2 hours at room temperature. Antigen retrieval was performed in 0.1M sodium citrate buffer and detected using Diaminobenzidine (DAB). Mouse IgG2b AT10F1 Purified Hu E, ICC/IF, P, WB
0.1 ml / €310.00
  Acris Antibodies GmbH

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