Phospho-RB1 (Ser795) Antibody

What is the biological significance of RB1 phosphorylation at Ser795?

The retinoblastoma (RB1) protein is a key regulator of cell cycle progression that acts as a tumor suppressor. In its underphosphorylated (active) form, RB1 interacts with E2F1 transcription factors and represses their activity, leading to cell cycle arrest . Phosphorylation at Ser795 is one of several phosphorylation events that inactivate RB1, releasing E2F1 and allowing cell cycle progression.

Specifically, Ser795 phosphorylation:

Proper storage and handling are crucial for maintaining antibody activity:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze/thaw cycles as they can denature and reduce antibody efficiency

• When stored as supplied (typically in phosphate buffered saline with glycerol and sodium azide), the antibody remains stable for at least 12 months

• For short-term use, aliquot upon delivery and keep at 4°C

• Working dilutions should be prepared fresh before use for optimal results

How can I validate the specificity of Phospho-RB1 (Ser795) Antibody in my experiments?

Validating antibody specificity is critical for reliable results:

• Positive and negative controls:

  • Use cell lines with known RB1 phosphorylation status (e.g., 293 cells or K-562 cells as shown in validation data)

  • Include phosphatase-treated samples as negative controls

• Phosphorylation induction:

  • Treat cells with ATP (5 mM at 30°C for 1 hour) to induce phosphorylation

  • Use serum starvation followed by serum stimulation to observe cell cycle-dependent phosphorylation changes

• Peptide competition:

  • Pre-incubate the antibody with the phosphorylated peptide used as immunogen

  • Signal should be blocked with the phospho-peptide but not with the non-phosphorylated version

• Phosphorylation site mutation:

  • Express wild-type and S795A mutant RB1 constructs

  • The antibody should detect only the wild-type protein when phosphorylated

What are the optimal lysis conditions for preserving RB1 phosphorylation status?

To accurately detect phosphorylated RB1 (Ser795), phosphorylation status must be preserved:

• Buffer composition:

  • Use buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Include protease inhibitors to prevent protein degradation

  • RIPA or NP-40 based buffers are generally suitable (e.g., PBS with 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)

• Sample handling:

  • Work quickly and keep samples on ice

  • Avoid repeated freeze/thaw cycles

  • Process samples immediately after collection

• Protein quantification:

  • Normalize loading to 25μg protein per lane for Western blotting as used in validation studies

  • Use BSA as a blocking agent (3% BSA recommended based on validation data)

How does RB1 Ser795 phosphorylation correlate with other phosphorylation sites on RB1?

RB1 contains multiple phosphorylation sites that work in concert to regulate its function:

Understanding the phosphorylation pattern at multiple sites provides greater insight into the cell cycle stage and the specific signaling pathways active in your experimental system. While Ser795 phosphorylation is an important marker, analyzing multiple sites simultaneously through multiplexed Western blotting or mass spectrometry provides more comprehensive information about RB1 regulatory status.

What approaches can I use to quantify changes in RB1 Ser795 phosphorylation in response to treatments?

Several quantitative approaches can be employed:

• Quantitative Western blotting:

  • Use dual detection with total RB1 antibody to normalize phospho-signal

  • Employ chemiluminescence detection with standard curves of recombinant phosphorylated proteins

  • Use digital imaging systems with broad dynamic range

• ELISA-based approaches:

  • Cell-based ELISA allows for higher throughput analysis

  • Normalize phospho-signal to total protein using dual-antibody detection systems

  • Standardize with positive control lysates

• Phospho-flow cytometry:

  • Enables single-cell analysis of phosphorylation status

  • Can be combined with cell cycle markers for correlative analysis

  • Requires optimization of fixation and permeabilization conditions

• Image-based quantification:

  • Use immunofluorescence or IHC with image analysis software

  • Enables spatial information about phosphorylation patterns

  • Can be normalized to total RB1 through dual staining approaches

How can I distinguish between non-specific signals and true Phospho-RB1 (Ser795) detection in Western blotting?

Non-specific signals can complicate interpretation of phospho-specific Western blots:

• Blocking optimization:

  • Use 3% BSA rather than milk as a blocking agent, as milk contains phosphoproteins that can interfere

  • Test different blocking conditions (duration, temperature, buffer composition)

• Antibody validation controls:

  • Include lysates from RB1-null cells as negative controls

  • Use competing phosphopeptides to confirm signal specificity

  • Compare multiple phospho-RB1 antibodies recognizing different epitopes

• Sample preparation refinements:

  • Prevent degradation by using fresh samples and appropriate inhibitors

  • Consider immunoprecipitation before Western blotting for enrichment of RB1

  • Optimize SDS-PAGE conditions for better separation (use 6-8% gels for the 110 kDa RB1 protein)

• Detection system considerations:

  • Use highly specific secondary antibodies

  • Optimize exposure times to avoid saturation

  • Consider using fluorescent secondaries for more quantitative analysis

Why might I see weak or no phospho-RB1 (Ser795) signal in Western blotting?

Several factors can contribute to weak phospho-RB1 signals:

• Cell cycle phase:

  • RB1 phosphorylation is cell cycle-dependent; synchronize cells or use proliferating populations

  • Serum-starved cells will have minimal phosphorylation

• Phosphatase activity:

  • Inadequate phosphatase inhibition during lysis can lead to signal loss

  • Include both serine/threonine and tyrosine phosphatase inhibitors

  • Keep samples cold throughout processing

• Technical factors:

  • Insufficient antibody concentration (try 1:500 dilution if 1:1000 is not working)

  • Inadequate transfer of high molecular weight proteins (110 kDa)

  • Inefficient antigen retrieval for fixed samples

• Biological factors:

  • Some cell types have naturally low levels of RB1 or phospho-RB1

  • Certain treatments or genetic backgrounds may affect RB1 expression or phosphorylation

What are effective strategies for multiplexing Phospho-RB1 (Ser795) with other antibodies?

Multiplexing allows simultaneous detection of multiple targets:

• Sequential immunoblotting:

  • Strip and reprobe membranes (use mild stripping buffers for phospho-epitopes)

  • Document complete stripping before reprobing

  • Consider species and isotype differences when selecting antibodies

• Fluorescent multiplexing:

  • Use secondary antibodies with distinct fluorophores

  • Ensure primary antibodies are from different host species

  • Optimize signal acquisition to prevent bleed-through

• Considerations for phospho-epitopes:

  • Start with phospho-specific antibodies before total protein detection

  • Use antibodies from different host species when detecting multiple phosphorylation sites

  • Validate signal specificity for each antibody individually before multiplexing

How can Phospho-RB1 (Ser795) antibody be used to evaluate CDK inhibitor efficacy?

CDK inhibitors are important cancer therapeutics that target the RB1 pathway:

• Monitoring treatment response:

  • Decreased Ser795 phosphorylation indicates CDK4/6 inhibitor efficacy

  • Time-course experiments reveal kinetics of dephosphorylation

  • Combine with cell cycle analysis to correlate with G1 arrest

• Experimental approach:

  • Treat cells with inhibitor dose series (typically 0.01-10 μM)

  • Harvest at multiple timepoints (6, 12, 24, 48 hours)

  • Normalize phospho-RB1 to total RB1 and quantify dose-response

• Predictive biomarker applications:

  • Profile cell lines for baseline Ser795 phosphorylation status

  • Correlate with sensitivity to CDK inhibitors

  • Identify potential resistance mechanisms when phosphorylation persists despite treatment

What approaches should I use for studying Phospho-RB1 (Ser795) in tissue samples?

Analyzing phospho-RB1 in tissues requires special considerations:

• Tissue preparation:

  • Use phosphatase inhibitors during tissue harvesting

  • Fix tissues rapidly (preferably less than 30 minutes post-collection)

  • For IHC, use high-pressure antigen retrieval with 10 mM citrate buffer pH 6.0

• Staining optimization:

  • Dilution range for IHC: 1:50-1:200

  • Include positive control tissues (e.g., proliferating tissues like breast cancer)

  • Use phosphatase-treated serial sections as negative controls

• Quantification approaches:

  • Score phospho-RB1 positivity in relation to total cell count

  • Correlate with proliferation markers (Ki-67, PCNA)

  • Consider digital pathology for unbiased quantification

• Validation examples:

  • The antibody has been validated on human breast cancer, mouse lung, and rat kidney tissues

  • Nuclear staining pattern is expected for phospho-RB1

  • Compare with total RB1 staining on serial sections

How do different CDKs contribute to RB1 Ser795 phosphorylation and how can this be experimentally determined?

Understanding the kinase specificity provides insight into cell cycle regulation:

• Kinase contributions:

  • CDK4/6-cyclin D complexes are primary kinases for Ser795

  • CDK2-cyclin E may contribute under certain conditions

  • Other CDKs may have compensatory roles in specific contexts

• Experimental approaches:

  • Use selective CDK inhibitors (palbociclib for CDK4/6, roscovitine for CDK2)

  • Generate kinase dead mutants or siRNA knockdowns

  • In vitro kinase assays with recombinant proteins

• Correlation with cell cycle:

  • Synchronize cells and analyze phosphorylation throughout cell cycle phases

  • Use dual staining with cell cycle markers

  • Compare with other RB1 phosphorylation sites with known kinase specificity

How can Phospho-RB1 (Ser795) antibody be used in single-cell analysis techniques?

Single-cell approaches provide new insights into cell-to-cell variability:

• Single-cell Western blotting:

  • Microfluidic platforms allow protein analysis from individual cells

  • Can correlate phospho-RB1 with other signaling proteins

  • Reveals heterogeneity masked in population-level studies

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies enable high-parameter single-cell analysis

  • Can simultaneously examine multiple phosphorylation sites

  • Requires validation of antibody performance after metal conjugation

• Imaging mass cytometry:

  • Combines spatial information with single-cell resolution

  • Can map phospho-RB1 distribution in heterogeneous tissues

  • Allows correlation with tissue architecture and microenvironment

What are the implications of RB1 Ser795 phosphorylation beyond cell cycle control?

Emerging research suggests broader roles for phosphorylated RB1:

• Chromatin regulation:

  • Phosphorylated RB1 affects histone methylation patterns

  • Influences constitutive heterochromatin stability

  • Controls histone H4 'Lys-20' trimethylation

• DNA damage response:

  • Phosphorylation status changes in response to genotoxic stress

  • May influence DNA repair pathway choice

  • Serves as a node connecting cell cycle and DNA damage networks

• Metabolic regulation:

  • Phosphorylated RB1 influences metabolic enzyme expression

  • May coordinate cell growth with cell division

  • Creates potential connections between oncogenic signaling and metabolic reprogramming

By understanding these diverse functions, researchers can design more comprehensive experiments to explore RB1's roles beyond traditional cell cycle control.