AGFG1 Antibody

What is AGFG1 and why is it significant in research?

AGFG1 (ArfGAP with FG repeats 1), also known as HIV-1 Rev Binding Protein (HRB), is a 58-62 kDa protein related to nucleoporins that mediates nucleocytoplasmic transport. Its significance lies in several key functions: it binds the activation domain of HIV-1 Rev protein when assembled onto its RNA target, facilitates the nuclear export of Rev-directed RNAs (essential for HIV-1 replication), plays a role in vesicle docking and fusion during acrosome biogenesis, and participates in clathrin-mediated endocytosis of select cargo . AGFG1 is also implicated in airway responsiveness, making it relevant for asthma research .

What types of AGFG1 antibodies are available for research applications?

Multiple AGFG1 antibodies are available, primarily polyclonal antibodies raised in rabbits. These antibodies target different regions of the protein:

• Antibodies targeting the N-terminal region (AA 1-30)

• Antibodies targeting the middle region

• Antibodies specific to AA 1-280

• Antibodies recognizing AA 179-228

• Antibodies against AA 276-380

Most commercial antibodies are unconjugated, though some FITC-conjugated options exist . The majority are validated for Western blotting, with many also tested for immunohistochemistry, immunofluorescence, and immunoprecipitation .

What are the typical reactivity profiles of AGFG1 antibodies?

Most AGFG1 antibodies show reactivity with human samples, with many cross-reacting with mouse and rat tissues . Some antibodies demonstrate broader cross-reactivity with species including rabbit, cow, dog, guinea pig, horse, chicken, monkey, and pig . These reactivity profiles are typically determined through validated testing in Western blot, immunohistochemistry, and other applications. When selecting an antibody, researchers should carefully review the validated species reactivity data for their specific application .

What are the optimal dilutions and conditions for Western blot applications with AGFG1 antibodies?

The recommended dilutions for Western blotting vary by manufacturer and specific antibody:

• 1:500-1:2000 is common for most products

• 1:500-1:1000 for some products

• 0.04-0.4 μg/mL for concentration-based recommendations

What are the recommended protocols for immunohistochemistry using AGFG1 antibodies?

For immunohistochemistry applications, recommended dilutions typically range from 1:20-1:200 . AGFG1 antibodies have been validated on human tissues including placenta, breast cancer tissue, and tonsil . Some protocols suggest antigen retrieval with TE buffer pH 9.0, though citrate buffer pH 6.0 may be used as an alternative . AGFG1 typically shows strong membranous positivity in trophoblastic cells when staining placenta tissue . For optimal results, researchers should follow manufacturer-specific protocols, as some antibodies may require specific antigen retrieval methods or detection systems .

How should AGFG1 antibodies be stored and handled to maintain reactivity?

Most AGFG1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . The recommended storage temperature is -20°C, where they typically remain stable for 12 months after shipment . Repeated freeze-thaw cycles should be avoided to maintain antibody integrity . Some manufacturers indicate that aliquoting is unnecessary for -20°C storage . Upon receiving antibodies shipped with ice packs, immediate storage at the recommended temperature is advised . Most suppliers do not recommend recycling antibodies after use as the buffer system changes and storage conditions for recycled antibodies may vary, potentially compromising performance efficiency .

How can I validate the specificity of AGFG1 antibodies in my experimental system?

To validate specificity, consider the following approaches:

• siRNA-mediated knockdown: This has been demonstrated effective for AGFG1 antibody validation. Decrease in signal upon siRNA-mediated knockdown confirms antibody specificity to AGFG1 .

• Multiple detection methods: Validate your findings using different applications (WB, IHC, IF) with the same antibody to ensure consistent protein detection .

• Cross-validation with different antibodies: Use antibodies targeting different epitopes of AGFG1 to confirm consistent detection patterns .

• Positive control selection: Use validated positive control samples such as mouse testis tissue or HeLa cells, which consistently show AGFG1 expression .

• Western blot band pattern analysis: Be aware of the expected 58-60 kDa band and note any secondary bands that may appear at ~47kDa, ~38kDa, or ~115kDa in certain cell lines .

What are the functional implications of AGFG1 in HIV-1 research, and how can antibodies advance this field?

AGFG1 plays a critical role in HIV-1 replication by facilitating the export of viral RNAs. When AGFG1's function is impaired via RNA interference or expression of a dominant negative mutant, intron-containing viral RNAs become sequestered at the perinuclear region . AGFG1 acts as a cofactor for viral Rev and promotes movement of Rev-responsive element-containing RNAs from the nuclear periphery to the cytoplasm, a step essential for HIV-1 replication .

Antibodies against AGFG1 can advance HIV-1 research through:

• Studying AGFG1 localization during viral infection using immunofluorescence

• Analyzing AGFG1 expression levels in different cell types to understand susceptibility to HIV-1

• Co-immunoprecipitation studies to investigate AGFG1 interactions with viral and cellular proteins

• Functional blocking studies to further elucidate AGFG1's role in viral RNA export

• Investigating AGFG1 as a potential therapeutic target for HIV-1 infection

The fruit fly homolog Drongo (Drosophila neural GTS1-like ORF) provides an additional model system for studying AGFG1's functions in RNA transport .

What is known about AGFG1's role in acrosome biogenesis and vesicular transport, and how can antibodies help investigate these functions?

AGFG1 is essential for vesicle docking and fusion during acrosome biogenesis in spermatogenesis . Unlike its role in HIV-1 replication, AGFG1 appears dispensable for cellular viability . In mammalian cells, AGFG1 localizes primarily at the perinuclear region and plasma membrane, but also throughout the cytoplasm, and participates in clathrin-mediated endocytosis of select cargo .

Researchers can use AGFG1 antibodies to:

• Study AGFG1 localization during spermatogenesis through immunohistochemistry and immunofluorescence

• Investigate protein interactions through co-immunoprecipitation to identify binding partners in vesicular transport pathways

• Examine AGFG1 expression patterns in different tissues to understand tissue-specific functions

• Analyze potential post-translational modifications that might regulate AGFG1's function in vesicular transport

• Explore the relationship between AGFG1's roles in RNA transport and vesicular trafficking

How can AGFG1 antibodies be used to investigate its potential role in asthma and airway responsiveness?

AGFG1 variants have been associated with severity of airway responsiveness (AHR), a primary characteristic of asthma . A genome-wide association study identified SNPs in/near AGFG1 that had nominally significant unadjusted P-values for being associated with expression levels of AGFG1 in whole blood, though these did not pass Benjamini-Hochberg correction criteria .

Researchers investigating AGFG1's role in asthma can use antibodies to:

• Compare AGFG1 expression levels in normal versus asthmatic airway tissues

• Examine AGFG1 localization in airway smooth muscle cells using immunohistochemistry and immunofluorescence

• Investigate potential changes in AGFG1 expression following exposure to asthma-related stimuli

• Study AGFG1 interactions with proteins involved in airway smooth muscle contractility through co-immunoprecipitation

• Analyze the effects of AGFG1 knockdown or overexpression on airway responsiveness in cellular or animal models

What are the comparative specifications of commercially available AGFG1 antibodies?

This table provides a comparative overview of specifications for researchers to select the most appropriate antibody for their specific experimental requirements .

What information should be included in Materials and Methods sections when reporting research using AGFG1 antibodies?

When reporting research using AGFG1 antibodies, include the following information in the Materials and Methods section:

• Antibody specifications:

  • Manufacturer and catalog number

  • Host species, clonality, and isotype

  • Target epitope or immunogen region if known

  • RRID (Research Resource Identifier) when available

• Application-specific details:

  • Dilution used for each application

  • Incubation conditions (time, temperature)

  • Detection method (e.g., HRP-conjugated secondary antibody)

  • Antigen retrieval method for IHC (buffer type, pH, conditions)

• Sample preparation:

  • Protein extraction method for Western blot

  • Fixation method for immunohistochemistry/immunofluorescence

  • Blocking reagents and conditions

• Controls employed:

  • Positive controls (e.g., mouse testis tissue, HeLa cells)

  • Negative controls (primary antibody omission, isotype controls)

  • Validation methods (e.g., siRNA knockdown)

• Image acquisition parameters:

  • Equipment specifications

  • Exposure settings

  • Software used for analysis