DNAJB12 Antibody

What is DNAJB12 and what cellular functions does it perform?

DNAJB12 belongs to the evolutionarily conserved DNAJ/HSP40 family of proteins, which regulate molecular chaperone activity by stimulating ATPase activity. It acts as a co-chaperone with HSPA8/Hsc70 and is required to promote protein folding and trafficking, prevent aggregation of client proteins, and promote unfolded proteins to the endoplasmic reticulum-associated degradation (ERAD) pathway .

In addition to its co-chaperone role, DNAJB12 can act independently of HSPA8/Hsc70: together with DNAJB14, it functions as a chaperone that promotes maturation of potassium channels KCND2 and KCNH2 by stabilizing nascent channel subunits and assembling them into tetramers. While stabilization of nascent channel proteins depends on HSPA8/Hsc70, the oligomerization of channel subunits occurs independently of HSPA8/Hsc70 .

DNAJB12 has also been implicated in viral infection processes, particularly in polyomavirus endoplasmic reticulum membrane penetration and infection .

What types of DNAJB12 antibodies are available for research applications?

Several types of DNAJB12 antibodies are available for research applications, with variations in host species, clonality, immunogen, and validated applications:

Most commonly available DNAJB12 antibodies are rabbit polyclonal antibodies, targeting different regions of the protein . The choice of antibody should be guided by the specific research application and species of interest.

What are the standard applications for DNAJB12 antibodies in cellular research?

DNAJB12 antibodies have been validated for several research applications, each with specific methodological considerations:

• Western Blotting (WB): All the reviewed antibodies are suitable for western blotting. For example, ab154410 has been successfully used at a 1/1000 dilution with various cell lysates including A549, H1299, HCT116, and MCF7, with the predicted band size of 41 kDa .

• Immunohistochemistry on Paraffin-embedded tissues (IHC-P): Some antibodies like ab154410 have been validated for IHC-P, used at a 1/500 dilution on human colon carcinoma tissue .

• Immunoprecipitation (IP): Antibodies such as A13448-1 from Boster Bio are suitable for immunoprecipitation studies, which can be valuable for studying DNAJB12's interactions with other proteins .

• ELISA: Several antibodies are validated for ELISA applications, allowing for quantitative analysis of DNAJB12 levels in various samples .

The choice of application depends on the specific research question, with western blotting being the most commonly validated technique across the different antibodies.

How can I optimize Western blot protocols specifically for DNAJB12 detection?

For optimal Western blot detection of DNAJB12, researchers should consider this methodological approach:

• Sample Preparation:

  • Use whole cell lysates from appropriate cell lines (A549, H1299, HCT116, MCF7 have been validated)

  • Load approximately 30 μg of protein per lane

  • Use 10% SDS-PAGE for optimal separation (DNAJB12 has a predicted molecular weight of 41-42 kDa)

• Antibody Incubation:

  • Primary antibody dilution: 1/1000 for ab154410 (other antibodies may require different dilutions)

  • Incubate overnight at 4°C for optimal binding

  • Use appropriate HRP-conjugated secondary antibody against rabbit IgG

• Controls and Validation:

  • Include positive controls like A549 or HCT116 cell lysates

  • If possible, include lysates from DNAJB12 knockdown cells as negative controls

  • Look for a specific band at the predicted molecular weight (41 kDa)

When troubleshooting, remember that membrane proteins like DNAJB12 may require specialized lysis buffers with appropriate detergents to ensure complete extraction from the ER membrane.

What methods are effective for studying DNAJB12's role in protein quality control pathways?

To investigate DNAJB12's role in protein quality control pathways, particularly in ERAD, researchers can employ these methodological approaches:

• RNA interference approach:

  • Knock down DNAJB12 using shRNA (as demonstrated in published studies)

  • Assess the impact on ERAD substrate degradation

  • Measure half-lives of known ERAD substrates with and without DNAJB12

• Co-immunoprecipitation (Co-IP) with HSPA8/Hsc70:

  • Use antibodies against DNAJB12 for IP

  • Western blot for HSPA8/Hsc70 and other known ERAD components

  • This helps identify direct protein interactions in the ERAD pathway

• Overexpression studies:

  • Express HA-tagged DNAJB12 in cells

  • Monitor effects on ERAD substrate levels

  • Assess changes in cellular stress responses

• Subcellular localization studies:

  • Use immunofluorescence to track DNAJB12 localization

  • Co-localize with ER markers and ERAD components

  • Assess changes in localization under different cellular stresses

These methodologies provide complementary data sets to understand how DNAJB12 functions within the broader context of cellular protein quality control.

What techniques can I use to investigate the interaction between DNAJB12 and DNAJB14?

To study the functional interaction between DNAJB12 and DNAJB14, researchers can employ the following methodological approaches:

• Confocal microscopy for co-localization:

  • Express tagged versions of both proteins (e.g., HA-tagged DNAJB12)

  • Perform immunofluorescence and analyze co-localization patterns

  • Look for structures like DJANGOS (DNA J-associated nuclear globular structures) that form when these proteins are overexpressed

• Knockdown and rescue experiments:

  • Knock down DNAJB12 and assess if DNAJB14 overexpression can rescue phenotypes

  • Research has shown that DNAJB14 requires DNAJB12 to induce DJANGOS, while DNAJB12 can induce DJANGOS without DNAJB14

  • This helps establish dependency relationships between the proteins

• Functional assays for potassium channel maturation:

  • Both proteins promote maturation of potassium channels KCND2 and KCNH2

  • Measure channel activity in cells with various combinations of DNAJB12/DNAJB14 expression

  • This helps understand their cooperative functions in channel biogenesis

• Electron microscopy:

  • Perform immunoEM to visualize subcellular structures like DJANGOS

  • This provides insight into how these proteins cooperate to form membranous structures

These techniques collectively provide a comprehensive understanding of the functional relationship between these co-chaperones.

How can I study DJANGOS formation in cells expressing DNAJB12?

DJANGOS (DNA J-associated nuclear globular structures) form when DNAJB12 or DNAJB14 is overexpressed. To study this phenomenon effectively, researchers can use these methodological approaches:

• Expression system optimization:

  • Generate concentrated retroviral stocks expressing HA-tagged DNAJB12

  • Infect target cells (HeLa, CV1, or human primary foreskin fibroblasts have been validated)

  • This approach typically results in 5-35% of cell nuclei displaying DJANGOS in different experiments

• Immunofluorescence visualization:

  • Fix cells and perform indirect immunofluorescence for the HA tag

  • Use confocal microscopy to visualize the punctate nuclear structures

  • Counterstain with DAPI to confirm nuclear localization

• Dependency studies:

  • Establish cell lines with reduced DNAJB12 or DNAJB14 expression using shRNAs

  • Overexpress the other protein to test dependency relationships

  • Research has shown that DNAJB14 requires DNAJB12 for DJANGOS formation, but not vice versa

• Ultrastructural analysis:

  • Perform immunoEM to visualize the fine structure of DJANGOS

  • This confirms that the structures seen by immunofluorescence correspond to membranous structures within nuclei

Understanding DJANGOS formation may provide insights into how these proteins function beyond their classical roles in the ER.

Why might I observe non-specific binding with my DNAJB12 antibody and how can I address it?

Non-specific binding with DNAJB12 antibodies can be addressed through these methodological solutions:

• Antibody validation with appropriate controls:

  • Use lysates from cells with confirmed DNAJB12 expression (e.g., A549, HCT116)

  • Include lysates from DNAJB12 knockdown cells as negative controls

  • Compare band patterns to identify true DNAJB12 signal (predicted at 41-42 kDa)

• Blocking optimization:

  • Test different blocking agents (BSA vs. non-fat milk)

  • Increase blocking time or concentration

  • Add 0.1-0.3% Tween-20 to reduce non-specific binding

• Antibody dilution titration:

  • Test a range of primary antibody dilutions (start with manufacturer recommendations, e.g., 1:1000 for ab154410)

  • Titrate secondary antibody to minimize background

• Consider epitope accessibility:

  • Different antibodies target different epitopes of DNAJB12

  • Some target aa 50 to C-terminus , others target aa51-100

  • The optimal antibody may depend on the sample preparation method and experimental conditions

Non-specific binding is often application-specific, so optimization strategies may need to be tailored to whether you're performing Western blotting, immunohistochemistry, or other techniques.

How can I address inconsistent DNAJB12 detection across different cell lines?

When facing inconsistent DNAJB12 detection across cell lines, researchers can implement these methodological approaches:

• Cell-type specific extraction optimization:

  • Different cell lines may require different lysis buffers

  • For membrane proteins like DNAJB12, ensure adequate solubilization with appropriate detergents

  • Include appropriate protease inhibitors to prevent degradation

• Loading optimization:

  • Different cell lines may express DNAJB12 at varying levels

  • Load more protein from cells with lower expression (up to 30 μg has been validated)

  • Use housekeeping proteins appropriate for the cell types being compared

• Antibody selection based on species reactivity:

  • Some antibodies show better cross-species reactivity than others

  • For example, Boster Bio's A13448-1 reacts with human, mouse, and rat DNAJB12

  • Antibodies-online's ABIN6744594 is optimized for mouse and rat samples

• Consider post-translational modifications:

  • DNAJB12 may undergo different post-translational modifications in different cell types

  • These modifications might affect antibody recognition

  • Use antibodies targeting different epitopes to address this issue

These strategies should help achieve more consistent detection across experimental conditions and cell types.

How can I investigate DNAJB12's role in viral infection pathways?

To study DNAJB12's role in viral infection, particularly with polyomaviruses like SV40, researchers can employ these methodological approaches:

• RNA interference to modulate DNAJB12 expression:

  • Use shRNAs targeting DNAJB12 to reduce its expression

  • Research has shown that knockdown of DNAJB12 reduced SV40 infectivity by approximately 50-fold

  • This approach helps establish the requirement for DNAJB12 in viral infection

• Viral infection assays with DNAJB12 modulation:

  • Infect control and DNAJB12-depleted cells with SV40

  • Quantify infection efficiency through virus-expressed reporter genes

  • The specific block in infection occurs at the step of viral capsid exit from the ER lumen prior to nuclear entry

• Structure-function analysis:

  • Express wild-type or mutant forms of DNAJB12 in knockdown cells

  • Test which domains of DNAJB12 are required for supporting viral infection

  • This helps identify the specific mechanisms by which DNAJB12 facilitates infection

• Co-localization studies during infection:

  • Track DNAJB12 localization during different stages of viral infection

  • Co-stain for viral proteins to identify potential interaction points

  • Use confocal microscopy to visualize these interactions

Understanding how DNAJB12 facilitates viral infection may reveal novel therapeutic targets for viral diseases.

What methods are most effective for studying DNAJB12's role in potassium channel maturation?

To investigate DNAJB12's role in potassium channel maturation, particularly for KCND2 and KCNH2 channels, researchers can implement these methodological approaches:

• Co-expression studies in heterologous systems:

  • Express potassium channels (KCND2 or KCNH2) with or without DNAJB12

  • Measure channel protein levels, cellular localization, and assembly into tetramers

  • Research shows DNAJB12 (with DNAJB14) promotes channel maturation by stabilizing nascent channel subunits and facilitating tetramer assembly

• Separation of HSPA8/Hsc70-dependent and independent functions:

  • Research indicates stabilization of nascent channel proteins is HSPA8/Hsc70-dependent, while oligomerization is HSPA8/Hsc70-independent

  • Use HSPA8/Hsc70 inhibitors or mutants to distinguish these functions

  • This helps delineate the specific mechanisms of DNAJB12 action

• Electrophysiological recordings:

  • Perform patch-clamp recordings to assess functional channel activity

  • Compare current amplitude and kinetics in cells with normal or altered DNAJB12 levels

  • This directly measures the functional outcome of DNAJB12's chaperone activity

• Pulse-chase analysis of channel biogenesis:

  • Label newly synthesized channel proteins and track their maturation

  • Compare half-lives and processing in control versus DNAJB12-depleted cells

  • Determine if DNAJB12 affects synthesis, degradation, or assembly of channels

These approaches provide complementary data on how DNAJB12 contributes to ion channel maturation, which may have implications for channelopathies and related disorders.