PGRMC1 Antibody

How do I select the appropriate PGRMC1 antibody for my research?

When selecting a PGRMC1 antibody, consider several critical factors to ensure experimental success. First, determine your application requirements (Western blot, immunohistochemistry, immunofluorescence, etc.) as antibody performance varies significantly between applications. Research publications have successfully used rabbit polyclonal antibodies raised against amino acids 48-130 of human PGRMC1 for Western blotting at 1:750 dilution and immunofluorescence at 1:250 dilution . Second, verify species reactivity, as orthologous PGRMC1 proteins exist in canine, porcine, monkey, mouse and rat models . Third, review validation data including specificity testing through knockdown experiments or immunoprecipitation studies. Finally, examine epitope location, as antibodies targeting different regions may detect different PGRMC1 post-translational modifications. For sumoylation studies or detection of higher molecular weight PGRMC1 forms, ensure your antibody can recognize these modified versions .

What controls should I include when working with PGRMC1 antibodies?

Proper controls are essential for validating PGRMC1 antibody specificity and experimental reliability. At minimum, include a negative control by omitting the primary antibody in parallel samples to identify any non-specific binding from secondary antibodies . For Western blotting, include a loading control (e.g., GAPDH antibody at 1:4000 dilution) to confirm equal protein loading across samples . When studying PGRMC1 in specific cell types, consider using PGRMC1-depleted cells (via siRNA knockdown) as negative controls to verify antibody specificity. For neutralization experiments, pre-incubate the PGRMC1 antibody with purified PGRMC1 protein before application to demonstrate binding specificity . In co-localization studies with other proteins such as SUMO1, include single primary antibody controls to rule out non-specific signals . These comprehensive controls will significantly enhance the reliability and reproducibility of your PGRMC1 research findings.

What are the optimal protocols for PGRMC1 detection by Western blotting?

For optimal PGRMC1 detection by Western blotting, follow this evidence-based protocol: Lyse cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with protease and phosphatase inhibitors, keeping all procedures on ice . Determine protein concentration using a bicinchoninic acid assay kit and load 10 μg of total protein per lane. After separation by SDS-PAGE, transfer proteins to nitrocellulose membranes and block with 5% BSA or non-fat milk. Incubate membranes with rabbit polyclonal PGRMC1 antibody (1:750 dilution) overnight at 4°C, followed by anti-rabbit HRP-labeled secondary antibody (1:2000) . Detect PGRMC1 using enhanced chemiluminescence.

Note that PGRMC1 appears at approximately 22-25 kDa, but also exhibits higher molecular weight bands (>50 kDa) in many cell types due to post-translational modifications including sumoylation . After detection, strip and reprobe membranes with GAPDH antibody (1:4000) as a loading control. Cell density can affect PGRMC1 expression patterns, with high-density cultures showing increased 22 kDa band intensity and decreased >50 kDa bands .

How can I optimize immunofluorescence staining for PGRMC1 localization studies?

For optimal immunofluorescence detection of PGRMC1, begin by fixing cells in 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 in PBS for 7 minutes . Block non-specific binding with 5% normal goat serum in PBS for 1 hour at room temperature. Incubate samples overnight at 4°C with rabbit polyclonal anti-PGRMC1 antibody at 1:250 dilution in 0.1% BSA/PBS . After washing, detect primary antibody using Alexa Fluor 546-labeled anti-rabbit secondary antibody (1:800 dilution) . Counterstain nuclei with Hoechst 33342 and mount with anti-fade reagent.

For co-localization studies with other proteins (e.g., SUMO1), use a mixture of PGRMC1 antibody and the second target antibody (e.g., mouse anti-SUMO1 at 1:50 dilution), followed by appropriate species-specific secondary antibodies with distinct fluorophores . Capture images using fluorescence microscopy with appropriate filter sets. When analyzing PGRMC1 localization, note that cellular distribution varies with cell density and cell cycle stage, with higher nuclear localization observed in actively dividing cells (approximately 50% confluent) compared to higher density cultures where PGRMC1 appears predominantly cytoplasmic .

What techniques can detect PGRMC1 interactions with other proteins?

Several complementary techniques effectively detect PGRMC1 interactions with partner proteins. Co-immunoprecipitation is fundamental - using PGRMC1 antibodies to pull down protein complexes followed by Western blotting to detect interacting partners. Research has successfully demonstrated PGRMC1-SUMO1 interactions using this approach, revealing a specific approximately 100 kDa band detected by both PGRMC1 and SUMO1 antibodies .

In situ proximity ligation assay (PLA) provides superior visualization of protein interactions within intact cells. This technique has been effective for studying PGRMC1-SUMO1 interactions, generating fluorescent spots only when the proteins are in close proximity . PLA has also revealed interactions between PGRMC1 and both PGRMC2 and G3BP2 . The technique allows quantification of interaction intensity under different conditions, such as progesterone treatment.

Immunofluorescence co-localization combined with confocal microscopy provides spatial information about PGRMC1 interactions. This approach has demonstrated PGRMC1 and PGRMC2 co-localization in granulosa cells . For functional validation of interactions, researchers have successfully used siRNA knockdown of either partner or antibody-mediated disruption of the complex to assess biological consequences, revealing that disrupting the PGRMC1:PGRMC2 complex increases cell cycle entry .

How does PGRMC1 expression correlate with cancer progression and patient outcomes?

Mechanistically, PGRMC1 promotes multiple aspects of cancer pathophysiology. In GBM, PGRMC1 enhances proliferation, anchorage-independent growth, and invasive capacity of cancer cells . At the molecular level, PGRMC1 appears to function through interaction with Integrin beta-1 (ITGB1) and TCF 1/7, with ITGB1 and PGRMC1 levels correlating in GBM patient tissues . Additionally, PGRMC1 contributes to tumor-related inflammation by enhancing Interleukin-8 production in GBM cells and promoting neutrophil recruitment. The expression of PGRMC1 significantly correlates with the numbers of tumor-infiltrating neutrophils in GBM patient tissues, potentially explaining another mechanism by which PGRMC1 influences disease progression .

How does PGRMC1 affect cancer cell response to therapeutic agents?

PGRMC1 expression significantly influences cancer cell response to therapeutic agents, with emerging evidence suggesting dual roles depending on treatment type. In glioblastoma (GBM), high PGRMC1 expression renders cancer cells less susceptible to temozolomide (TMZ), the standard chemotherapeutic agent for this cancer type . This finding suggests that PGRMC1 may contribute to chemoresistance mechanisms, potentially by promoting survival pathways or DNA damage repair.

Intriguingly, PGRMC1 appears to have the opposite effect on sensitivity to ferroptosis inducers. GBM cells with high PGRMC1 expression show increased susceptibility to the ferroptosis inducer erastin . This differential effect on treatment response highlights PGRMC1 as a potential biomarker for predicting treatment efficacy and suggests novel therapeutic strategies for PGRMC1-high tumors. The mechanism behind this differential response may relate to PGRMC1's involvement in lipid metabolism and cellular redox balance, which are critical processes in ferroptotic cell death.

These findings collectively indicate that PGRMC1 assessment in tumor samples could guide personalized therapeutic approaches, potentially steering treatment away from standard chemotherapeutics and toward ferroptosis inducers in PGRMC1-high tumors.

What methodologies are most effective for studying PGRMC1's role in tumor-immune interactions?

Studying PGRMC1's role in tumor-immune interactions requires integrating multiple complementary methodologies. For clinical relevance, immunohistochemical co-staining of patient tumor sections for PGRMC1 and immune cell markers (particularly neutrophil markers) provides valuable correlation data. Research has demonstrated significant correlation between PGRMC1 expression and tumor-infiltrating neutrophils in GBM patient tissues .

In vitro, conditioned media experiments effectively assess how PGRMC1 levels in cancer cells affect immune cell recruitment and function. PGRMC1 enhances Interleukin-8 production in GBM cells, promoting neutrophil recruitment . To establish causality, stable PGRMC1 knockdown models in cancer cell lines, followed by assessment of cytokine/chemokine production profiles using multiplex ELISA or cytokine arrays, can identify the specific inflammatory mediators regulated by PGRMC1.

For mechanistic insights, co-culture systems combining PGRMC1-manipulated cancer cells with immune cells allow assessment of direct cell-cell interactions. Flow cytometry analysis of immune cell activation markers and functional assays measuring immune cell cytotoxicity, cytokine production, and migration provide comprehensive functional data. Additionally, in vivo models using PGRMC1-knockdown cancer cells implanted into immunocompetent mice enable assessment of tumor-immune interactions in a physiologically relevant context, with subsequent flow cytometry and immunohistochemical analysis of tumor-infiltrating immune cells.

How do PGRMC1 and PGRMC2 cooperatively regulate the cell cycle?

PGRMC1 and PGRMC2 function cooperatively to regulate multiple aspects of the cell cycle, particularly in granulosa cells. These proteins form a complex that controls cell cycle entry and progression through metaphase. Research in Spontaneously Immortalized Granulosa Cells (SIGCs) reveals that depleting either PGRMC1 or PGRMC2 increases entry into the cell cycle, specifically promoting G0 to G1 transition . This suggests the PGRMC1:PGRMC2 complex normally functions to maintain cells in G0, restraining cell cycle entry.

Mechanistically, pull-down assays, colocalization studies, and in situ proximity ligation assays (PLA) all confirm that PGRMC1 physically binds PGRMC2 . Disrupting this complex through siRNA-mediated depletion of either protein or through cytoplasmic delivery of a PGRMC2 antibody increases cell cycle entry, while overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion proteins inhibits entry into the cell cycle .

Beyond regulating cell cycle entry, both PGRMC1 and PGRMC2 localize to the mitotic spindle during cell division. Their absence disrupts normal mitosis, causing cells to arrest in metaphase and subsequently undergo apoptosis . This dual regulatory role—controlling both entry into the cell cycle and progression through mitosis—positions the PGRMC1:PGRMC2 complex as a master regulator of cellular proliferation in granulosa cells and potentially other cell types.

What experimental approaches best demonstrate PGRMC1's effect on cellular proliferation?

To comprehensively assess PGRMC1's effects on cellular proliferation, researchers should employ multiple complementary approaches. Gene knockdown studies using siRNA or CRISPR-Cas9 techniques provide critical insights. Research demonstrates that depleting PGRMC1 in Spontaneously Immortalized Granulosa Cells (SIGCs) increases entry into the cell cycle but paradoxically does not increase cell proliferation due to metaphase arrest and subsequent apoptosis .

Cell cycle distribution analysis using flow cytometry with propidium iodide staining or BrdU incorporation assays reveals specific cell cycle phases affected by PGRMC1 manipulation. Studies show that depleting PGRMC1 reduces the percentage of cells in G0 and increases the percentage in G1, demonstrating PGRMC1's role in regulating G0/G1 transition . For in vivo validation, conditional knockout models provide tissue-specific insights into PGRMC1's proliferative effects, as demonstrated in Pgrmc1 d/d mice compared to Pgrmc1 fl/fl controls .

Time-lapse microscopy of cells expressing fluorescent markers for cell cycle phases (e.g., FUCCI system) allows real-time tracking of individual cells through the cell cycle following PGRMC1 manipulation. Finally, rescue experiments re-introducing wild-type or mutant PGRMC1 into PGRMC1-depleted cells confirm causality and identify critical domains or post-translational modifications required for PGRMC1's proliferative effects.

How does cellular localization of PGRMC1 change during the cell cycle?

This subcellular redistribution correlates with changes in PGRMC1 molecular weight forms detected by Western blotting - specifically, higher molecular weight bands (>50 kDa) decrease while the approximately 22 kDa band increases as cells become confluent . This suggests post-translational modifications, likely sumoylation based on the presence of three conserved sumoylation sites in PGRMC1, regulate its localization and function throughout the cell cycle .

During mitosis, both PGRMC1 and PGRMC2 localize to the mitotic spindle, explaining why their depletion causes cells to arrest in metaphase . This spindle localization is consistent with PGRMC1's role in regulating mitotic progression. In granulosa cells of ovarian follicles, PGRMC1 shows both cytoplasmic and nuclear localization, with increased nuclear PGRMC1 apparent in early atretic follicles . These localization patterns provide valuable insights into PGRMC1's cell cycle regulatory functions.

How can post-translational modifications of PGRMC1 be effectively studied?

Studying PGRMC1 post-translational modifications requires an integrated approach combining multiple techniques. Western blotting using gradient gels (6-15%) can effectively separate multiple PGRMC1 forms, revealing both the ~22 kDa unmodified protein and higher molecular weight bands (>50 kDa) representing modified forms . When examining sumoylation specifically, co-immunoprecipitation assays using SUMO1 antibodies followed by PGRMC1 detection have successfully identified sumoylated PGRMC1 at approximately 100 kDa .

For visualizing modified PGRMC1 in intact cells, proximity ligation assays (PLA) offer superior sensitivity and specificity. This technique has been used effectively to detect PGRMC1-SUMO1 interaction in fixed cells, revealing discrete fluorescent spots that represent sites of interaction . The PLA approach allows quantitative assessment of how various stimuli, such as progesterone treatment, affect PGRMC1 modification levels .

Mass spectrometry analysis of immunoprecipitated PGRMC1 can comprehensively identify all modification sites and types. For functional studies, comparing wild-type PGRMC1 with site-directed mutants where specific modification sites are altered (e.g., lysine to arginine mutations at sumoylation sites) helps establish the biological significance of each modification. Finally, phospho-specific or SUMO-specific antibodies can detect specific modified forms of PGRMC1, though these may require custom development for optimal specificity.

What are the critical factors influencing PGRMC1 antibody specificity and sensitivity?

Multiple factors critically influence PGRMC1 antibody specificity and sensitivity. The epitope location is paramount—antibodies targeting different regions of PGRMC1 may preferentially detect specific post-translationally modified forms. For instance, antibodies raised against amino acids 48-130 of human PGRMC1 effectively detect both the ~22 kDa unmodified form and higher molecular weight (>50 kDa) modified forms .

Sample preparation significantly impacts detection quality—RIPA buffer effectively extracts PGRMC1 for Western blotting, but gentler lysis methods may better preserve protein-protein interactions for co-immunoprecipitation studies . Post-translational modifications affect epitope accessibility; PGRMC1 undergoes sumoylation and potentially other modifications that can mask antibody binding sites .

Fixation and permeabilization conditions are critical for immunohistochemistry and immunofluorescence—paraformaldehyde fixation (4%) followed by Triton X-100 permeabilization (0.1%, 7 minutes) works effectively for PGRMC1 detection in cultured cells . Antibody concentration requires optimization for each application: 1:750 dilution for Western blotting and 1:250 for immunofluorescence have been validated . Finally, detection methods influence sensitivity—enhanced chemiluminescence effectively visualizes PGRMC1 in Western blots, while fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 546) provide excellent sensitivity for immunofluorescence .

What approaches can resolve contradictory findings in PGRMC1 research?

Resolving contradictory findings in PGRMC1 research requires systematic investigation of several key variables. First, consider cell type and context differences—PGRMC1 function varies significantly between tissues and cell types, with distinct roles documented in cancer cells versus normal cells. Studies in granulosa cells versus glioblastoma cells might yield apparently contradictory results due to tissue-specific binding partners and signaling networks .

Post-translational modifications critically affect PGRMC1 function—apparent contradictions may arise when studies detect different PGRMC1 forms. The ~22 kDa unmodified protein may have different functions than higher molecular weight (>50 kDa) modified forms . Experimental conditions including cell density significantly impact PGRMC1 localization and molecular weight forms, with high-density cultures showing predominantly cytoplasmic PGRMC1 and increased 22 kDa form, while low-density cultures exhibit more nuclear PGRMC1 and increased >50 kDa forms .

Antibody selection introduces variability—antibodies targeting different epitopes may preferentially detect specific PGRMC1 forms or conformations. For definitive resolution of contradictions, integrate multiple approaches: validate key findings using both genetic (siRNA/CRISPR) and pharmacological approaches, confirm protein-protein interactions using complementary techniques (co-IP, PLA, co-localization), and directly compare experimental conditions from contradictory studies within the same experimental system.

How can I troubleshoot weak or absent PGRMC1 signal in Western blotting?

When experiencing weak or absent PGRMC1 signal in Western blotting, consider several evidence-based troubleshooting approaches. First, optimize protein extraction—PGRMC1 has been successfully extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) with protease and phosphatase inhibitors . Ensure all procedures are conducted on ice to preserve protein integrity.

Verify protein loading using a bicinchoninic acid assay and load adequate protein (10 μg per lane is typically sufficient) . PGRMC1 appears at multiple molecular weights—primarily at ~22 kDa but also at >50 kDa due to post-translational modifications—so examine the entire blot rather than focusing on a single molecular weight region . If the expected 22 kDa band is weak, check higher molecular weight regions for modified PGRMC1 forms.

Optimize antibody concentration; a 1:750 dilution of rabbit polyclonal anti-PGRMC1 has been validated . Extend primary antibody incubation to overnight at 4°C for maximum sensitivity. For detection, enhanced chemiluminescence with extended exposure times may be necessary for weakly expressed PGRMC1 . If signals remain weak, consider membrane stripping and reprobing with a different PGRMC1 antibody targeting an alternative epitope. Finally, verify PGRMC1 expression in your cell type by checking transcript levels via RT-qPCR, as expression varies significantly between cell types.

What factors affect reproducibility in PGRMC1 localization studies?

Multiple factors significantly impact reproducibility in PGRMC1 localization studies. Cell density critically influences PGRMC1 subcellular distribution—actively dividing cells at 50% confluence show both nuclear and cytoplasmic PGRMC1, while high-density cultures (72-96 hours) show predominantly cytoplasmic localization . Standardize cell seeding density and fixation timing to ensure consistent results.

Cell cycle stage dramatically affects PGRMC1 localization—cells in active mitosis show distinct PGRMC1 distribution patterns, particularly at the mitotic spindle . Consider synchronizing cells or co-staining for cell cycle markers to account for this variability. Fixation and permeabilization protocols significantly impact epitope accessibility—4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization for 7 minutes has been validated for PGRMC1 immunofluorescence .

Antibody selection introduces variability—antibodies targeting different PGRMC1 epitopes may preferentially detect specific subcellular pools or modified forms. The rabbit polyclonal antibody against amino acids 48-130 of human PGRMC1 at 1:250 dilution has been validated for immunofluorescence . Finally, image acquisition parameters dramatically affect perceived localization—standardize exposure times, gain settings, and post-acquisition processing. For accurate assessment of PGRMC1 nuclear/cytoplasmic distribution, capture images in multiple random fields using identical settings and perform quantitative analysis of signal intensity in each compartment.

What are the major sources of variability in PGRMC1-protein interaction studies?

Multiple factors contribute to variability in PGRMC1-protein interaction studies. Post-translational modifications significantly influence PGRMC1's interaction capacity—PGRMC1 undergoes sumoylation and potentially other modifications that alter its binding properties . The modification state varies with cell density and cell cycle stage, contributing to experimental variability.

Cell lysis conditions critically impact interaction preservation—harsh detergents may disrupt weak or transient interactions. For co-immunoprecipitation studies, gentler lysis buffers containing lower detergent concentrations better preserve protein complexes than RIPA buffer. Antibody selection introduces significant variability—antibodies targeting different PGRMC1 epitopes may differentially affect protein interactions or preferentially immunoprecipitate specific PGRMC1 complexes.

The biological context dramatically influences PGRMC1 interactions—hormonal treatments, particularly progesterone, affect PGRMC1-SUMO1 interaction as quantified by proximity ligation assay . Standardize culture conditions and treatments to minimize this variability. Technical factors in proximity ligation assays (PLA) affect sensitivity—optimization of antibody concentrations, incubation times, and washing conditions is essential for reproducible results .

To minimize variability, implement rigorous controls including IgG controls for co-immunoprecipitation, single primary antibody controls for PLA, and validation of key interactions using complementary approaches (co-IP, PLA, and co-localization studies). Additionally, quantify interaction intensity across multiple biological replicates using objective methods such as counting PLA spots per cell with automated image analysis software .