CHMP1B Antibody

What is CHMP1B and what cellular functions does it perform?

CHMP1B (Chromatin Modifying Protein 1B) is a component of the ESCRT-III (Endosomal Sorting Complex Required for Transport-III) machinery, playing crucial roles in multivesicular body biogenesis and receptor trafficking. It functions in the late stages of the endosomal sorting pathway and is involved in membrane deformation and scission processes. CHMP1B exists in different conformational states including monomers, dimers, and polymers, with its polymeric form participating in a ~500 kDa complex with other ESCRT-III proteins like IST1 . Its activity is critical for processes such as EGFR trafficking in human cells and during morphogenetic events like Drosophila wing development .

What are the molecular characteristics of CHMP1B protein?

CHMP1B has a calculated molecular weight of 22 kDa but is typically observed at approximately 28 kDa on SDS-PAGE gels . The protein contains multiple α-helical domains that mediate various protein-protein interactions. Notably, α-helices 4, 5, and 6 (residues 105-199) participate in interactions with proteins such as USP8 . CHMP1B can form SDS-resistant polymers detectable at approximately 200 kDa that can be disrupted by extended heat denaturation. Additionally, it contains critical lysine residues (particularly K87 and K90) that serve as ubiquitination sites regulating its function and subcellular localization .

What types of CHMP1B antibodies are available for research applications?

Several validated CHMP1B antibodies are available for research. The most extensively validated is Proteintech's 14639-1-AP, a rabbit polyclonal antibody referenced in at least 10 publications and tested for Western blot, ELISA, immunoprecipitation, and immunohistochemistry applications . This antibody shows reactivity with human, mouse, and rat samples. Other validated options include LSBio's LS-C482985 polyclonal antibody (for WB and IHC) and Invitrogen's version of the 14639-1-AP antibody that has been validated for multiple applications .

What are the recommended protocols and dilutions for using CHMP1B antibody in Western blot experiments?

For Western blot applications using CHMP1B antibody (such as 14639-1-AP), the recommended dilution range is 1:2000-1:12000 . When detecting endogenous CHMP1B, researchers should be aware that the protein typically appears in three distinct forms: monomers (~28 kDa), SDS-resistant dimers (~55 kDa), and polymers (~200 kDa) . The polymeric forms can be partially converted to dimeric forms with extended heat denaturation. For optimal results with recombinant GFP-tagged CHMP1B, note that the tag may interfere with polymerization, resulting primarily in detection of the monomeric form . Researchers should follow manufacturer protocols that typically include transfer to nitrocellulose or PVDF membranes, blocking with 5% non-fat milk or BSA, and overnight primary antibody incubation at 4°C.

How can CHMP1B antibodies be used effectively in immunoprecipitation studies?

For immunoprecipitation applications, CHMP1B antibody (14639-1-AP) should be used at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate . When studying interactions between CHMP1B and potential binding partners, it's important to note that endogenous CHMP1B immunoprecipitation with polyclonal antibodies (such as those recognizing residues 35-84) may preferentially pull down polymeric and ubiquitinated forms, but not non-ubiquitinated monomers. This selectivity occurs because the epitopes recognized by these antibodies may be masked in the closed conformation of monomeric CHMP1B . For interaction studies, researchers have successfully used both co-immunoprecipitation and complementary approaches like Venus complementation assays to verify CHMP1B interactions with proteins such as USP8 .

What tissue and sample types have been validated for CHMP1B antibody applications?

CHMP1B antibody (14639-1-AP) has been validated in multiple tissue types and cell lines. In Western blot applications, positive detection has been confirmed in A431 cells, human heart tissue, mouse heart tissue, rat heart tissue, and HeLa cells . For immunoprecipitation studies, HeLa cells have been successfully used. In immunohistochemistry applications, human kidney tissue has been validated, with recommended antigen retrieval using TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) . Published applications cite reactivity in both human and mouse samples for Western blot and immunofluorescence experiments .

How is CHMP1B regulated by ubiquitination and what role does USP8/UBPY play?

CHMP1B function and localization are dynamically regulated by ubiquitination and deubiquitination processes. Ubiquitination of CHMP1B can be induced within minutes of EGF stimulation, resulting in transient accumulation of ubiquitinated forms on membranes . Four lysine residues (particularly K87 and K90) within or close to a flexible loop of CHMP1B serve as ubiquitination sites, and mutation of these residues (CHMP1B-4K>R) leads to reduced ubiquitin linkage . USP8/UBPY, a deubiquitinating enzyme, directly interacts with CHMP1B via α-helices 4, 5, and 6 of CHMP1B and mediates its deubiquitination. This interaction occurs predominantly at late endosomes/lysosomes as demonstrated by co-localization with the Lamp1 marker . Notably, expression of wild-type or constitutively active USP8 (S680A), but not catalytically inactive USP8 (C748A), results in significant reduction of ubiquitinated CHMP1B pools, confirming USP8's role as a CHMP1B deubiquitinase .

What are the structural conformations of CHMP1B and how do they relate to function?

CHMP1B exists in at least three distinct conformational states: monomers (~28 kDa), dimers (~55 kDa), and polymers (~200 kDa) . The polymeric forms are SDS-resistant and participate in a ~500 kDa complex containing other ESCRT-III proteins such as IST1 . These polymers can be partially disrupted by extended heat denaturation. Importantly, the conformational state affects epitope accessibility - in monomeric CHMP1B, the auto-inhibitory helix 6 masks certain epitopes (such as those in the region of residues 35-84), preventing recognition by some antibodies in native conditions. These epitopes become exposed in polymeric and ubiquitinated forms . The functional significance of these different states relates to CHMP1B's role in membrane remodeling during multivesicular body formation, with the transition from closed monomers to open polymers being a key regulatory step in ESCRT-III function.

What is known about the CHMP1B interactome and how can researchers study these interactions?

CHMP1B interacts with multiple proteins as part of its function in the ESCRT pathway. A well-documented interaction partner is USP8/UBPY, which binds to α-helices 4, 5, and 6 (residues 105-199) of CHMP1B . This interaction has been verified through multiple approaches including co-immunoprecipitation, yeast two-hybrid, and Venus complementation assays. Another known interaction partner is IST1, which co-elutes with CHMP1B in a ~500 kDa complex as demonstrated through size exclusion chromatography . CHMP1B also interacts with Spastin through similar regions as its USP8 interaction. To study these interactions, researchers can employ co-immunoprecipitation using stringent conditions to minimize non-specific binding, Venus/BiFC complementation assays for visualizing interactions in living cells, or biochemical approaches like sucrose gradient separation followed by size exclusion chromatography to isolate and characterize native CHMP1B-containing complexes .

How can researchers distinguish between different molecular forms of CHMP1B in experimental systems?

When studying CHMP1B, researchers often encounter multiple molecular forms that can complicate data interpretation. To differentiate between monomers (~28 kDa), dimers (~55 kDa), and polymers (~200 kDa), several approaches can be employed: (1) Use gradient gels (6-12%) for better separation of the wide molecular weight range; (2) Vary sample denaturation conditions - extended heat denaturation (>10 minutes) can partially convert polymeric forms to dimers ; (3) For isolation of specific forms, employ sucrose gradient separation where polymers typically sediment in 20-30% sucrose fractions while dimers remain in 0-10% fractions ; (4) Use size exclusion chromatography to separate the ~500 kDa CHMP1B-containing complex from other forms; (5) For validation, employ CHMP1B knockdown controls using independent shRNAs to confirm band specificity . Note that recombinant GFP-tagged CHMP1B may predominantly appear as monomers due to the tag's interference with polymerization.

What are the common challenges in detecting ubiquitinated forms of CHMP1B?

Detecting ubiquitinated CHMP1B presents several technical challenges. For optimal results, researchers should: (1) Use highly stringent immunoprecipitation conditions to specifically detect ubiquitin moieties covalently linked to CHMP1B rather than interacting partners; (2) When co-expressing HA-tagged ubiquitin with GFP-CHMP1B, a major Ub-CHMP1B product typically migrates at ~70 kDa (representing mono- or di-ubiquitinated forms) ; (3) Be aware that CHMP1B appears to be modified by non-K48-linked ubiquitin chains, as K48-specific antibodies show minimal detection; (4) Accordingly, proteasome inhibitors do not significantly increase ubiquitinated CHMP1B levels ; (5) For studying endogenous ubiquitinated CHMP1B, use the FK2 anti-ubiquitin antibody for immunoprecipitation followed by CHMP1B immunoblotting; (6) Understand that only polymeric and ubiquitinated forms, not non-ubiquitinated monomers, are typically immunoprecipitated by antibodies recognizing residues 35-84 due to epitope masking in the closed conformation .

What controls should be included when studying CHMP1B ubiquitination and deubiquitination?

When investigating CHMP1B ubiquitination dynamics, several critical controls should be included: (1) For ubiquitination studies, compare wild-type CHMP1B with the CHMP1B-4K>R mutant (lysines 42, 59, 87, and 90 mutated to arginines) which shows substantially reduced ubiquitination ; (2) For more precise analysis, single lysine mutants (especially K87R and K90R) can help identify the primary ubiquitination sites ; (3) When studying USP8-mediated deubiquitination, compare wild-type USP8, constitutively active USP8 S680A, and catalytically inactive USP8 C748A - only the first two should reduce ubiquitinated CHMP1B levels ; (4) Include USP8 knockdown controls using siRNA, which should result in increased ubiquitinated CHMP1B ; (5) For specificity, use antibodies against different ubiquitin linkages (K48, K63, etc.); (6) To verify the absence of proteasomal degradation, include proteasome inhibitors like MG132 as controls; (7) For endogenous studies, validate antibody specificity using CHMP1B knockdown or knockout controls.

How should researchers interpret changes in CHMP1B ubiquitination in response to cellular stimuli?

When analyzing CHMP1B ubiquitination changes in response to stimuli such as EGF treatment, researchers should consider several key aspects: (1) Temporal dynamics - CHMP1B ubiquitination can be induced within minutes of EGF stimulation, so time-course experiments with appropriate sampling intervals are essential ; (2) Subcellular localization - ubiquitinated CHMP1B transiently accumulates on membranes, so fractionation approaches or imaging techniques might be needed to fully capture the changes ; (3) Functional significance - mutations in ubiquitination sites (CHMP1B-4K>R) render the protein non-functional in EGFR trafficking and Drosophila wing morphogenesis, suggesting ubiquitination is critical for CHMP1B function ; (4) Relationship to USP8 activity - as USP8 is activated by growth factor stimulation, the balance between ubiquitination and deubiquitination may represent a regulatory mechanism for CHMP1B; (5) Coordination with other ESCRT-III components - examine whether ubiquitination affects CHMP1B's interactions with partners like IST1 or influences the assembly/disassembly of the ~500 kDa complex.

What aspects of experimental design are crucial when comparing CHMP1B antibody results across different studies?

When comparing CHMP1B antibody data across studies, researchers should carefully consider: (1) Antibody epitope locations - antibodies recognizing different regions (particularly those binding to residues 35-84 versus other domains) may preferentially detect certain conformational states of CHMP1B ; (2) Sample preparation methods - denaturation conditions significantly affect the detection of polymeric versus monomeric forms; (3) Expression systems - endogenous CHMP1B typically displays three forms (monomers, dimers, polymers), while GFP-tagged CHMP1B may predominantly appear as monomers ; (4) Cell types and stimulation conditions - CHMP1B ubiquitination is inducible by EGF and potentially other stimuli, requiring careful timing of experiments; (5) Fractionation approaches - membrane-associated versus cytosolic CHMP1B may show different modification patterns; (6) Detection methods - different antibody dilutions (ranging from 1:50 for IHC to 1:12000 for WB) may be required for optimal results depending on the application ; (7) In immunoprecipitation experiments, the amount of antibody (0.5-4.0 μg per 1.0-3.0 mg lysate) can significantly affect results, requiring careful titration .

How can researchers integrate CHMP1B structural information with functional studies using antibodies?

To effectively integrate structural and functional analyses of CHMP1B, researchers should: (1) Consider the conformational states when designing experiments - the closed monomeric form may mask epitopes in residues 35-84 that become exposed in polymeric or ubiquitinated forms ; (2) When studying CHMP1B interactions (e.g., with USP8), target α-helices 4, 5, and 6 (residues 105-199), which have been shown to mediate protein-protein binding ; (3) For mutation studies affecting ubiquitination, focus on lysines 87 and 90 in the flexible loop region, which appear most critical for ubiquitin attachment ; (4) Use complementary approaches to verify structural states - combine biochemical fractionation (sucrose gradients, size exclusion chromatography) with imaging techniques (fluorescence microscopy with appropriate tagged constructs); (5) When studying membrane association, consider that ubiquitination affects CHMP1B membrane recruitment and function in EGFR trafficking ; (6) For comprehensive analysis, examine CHMP1B in the context of its ~500 kDa complex containing IST1 and potentially other ESCRT-III components ; (7) Validate functional impacts of structural or post-translational modifications using appropriate cellular or developmental models, as demonstrated by wing morphogenesis defects in Drosophila expressing CHMP1B-4K>R .