Phospho-RB1 (Ser780) Antibody

What is Phospho-RB1 (Ser780) and why is it significant in cell cycle research?

Phospho-RB1 (Ser780) refers to the retinoblastoma protein 1 (RB1) that has been phosphorylated at the serine 780 residue. RB1 is a 110 kDa tumor suppressor protein that functions as a key regulator of entry into cell division by acting as a transcriptional repressor of E2F1 target genes .

The phosphorylation status of RB1 is critical to its function:

• In its underphosphorylated (active) form, RB1 interacts with E2F1 and represses its transcription activity, leading to cell cycle arrest

• Phosphorylation at Ser780 is mediated by cyclin-dependent kinases (CDKs)

• Phosphorylation reduces RB1's affinity for E2F transcription factors, relieving transcriptional repression and allowing cell cycle progression from G1 to S phase

Research significance: Monitoring Ser780 phosphorylation provides insights into cell cycle regulation, cancer development, and cellular responses to various treatments. Cyclin D1 is specifically required for Ser780 phosphorylation in vivo, making this site a particularly important marker for cell cycle progression controlled by the cyclin D-CDK4/6 pathway .

What applications are recommended for Phospho-RB1 (Ser780) antibodies?

Phospho-RB1 (Ser780) antibodies can be used in multiple research applications, with varying recommended dilutions depending on the specific antibody:

When selecting an application, consider:

• Western blotting provides information about protein size and relative abundance

• IHC/ICC reveals spatial distribution within tissues/cells

• Flow cytometry allows quantitative single-cell analysis

• ELISA offers higher throughput quantification

Always optimize dilutions for your specific experimental conditions and sample types .

How should samples be prepared for optimal detection of Phospho-RB1 (Ser780)?

Proper sample preparation is crucial for maintaining phosphorylation states and achieving reliable results:

Lysis procedure:

• Prepare complete lysis buffer immediately prior to sample processing

• Use buffers containing phosphatase inhibitors to prevent dephosphorylation of RB1

• For cell lysates, rapid harvesting and immediate lysis minimizes phosphorylation changes

Protocol recommendations:

• Wash cells with ice-cold PBS before lysis

• Add protease and phosphatase inhibitor cocktails to lysis buffer

• Keep samples on ice during processing

• Clarify lysates by centrifugation (14,000g for 10 minutes at 4°C)

• Determine protein concentration using a compatible assay (e.g., BCA)

• Dilute samples to equivalent protein concentrations using complete lysis buffer

• For Western blotting, add reducing agent and denature by heating at 95°C for 5 minutes

Sample handling considerations:

• Flash-freeze tissue samples in liquid nitrogen to preserve phosphorylation status

• Store prepared samples at -80°C, avoid repeated freeze-thaw cycles

• For cell cycle studies, synchronize cells or use treatments like nocodazole (0.2 μg/mL for 18 hours) to enrich for specific cell cycle phases

What are appropriate controls when working with Phospho-RB1 (Ser780) antibodies?

Implementing proper controls is essential for experimental validity:

Positive controls:

• Nocodazole-treated cells (0.2 μg/mL for 18 hours) show increased phosphorylation at Ser780

• Lysates from rapidly proliferating cell lines (e.g., HT29 cells)

• Recombinant phosphorylated RB1 protein (if available)

Negative controls:

• λ phosphatase-treated samples to remove phosphorylation

• Tetrandrine-treated cells (30 μM, 18 hours) show decreased phosphorylation

• Contact-inhibited or serum-starved cells with minimal RB1 phosphorylation

• Primary antibody omission control

Specificity controls:

• Peptide competition assay using the phosphorylated immunogen peptide

• Parallel detection with total RB1 antibody to normalize phospho-signal

• Comparison between phospho-specific antibody signal and total RB1 in various treatment conditions

For quantitative applications, consider preparing a titration curve of positive and negative control lysates to establish assay dynamic range and sensitivity, as shown in this example:

This approach helps determine optimal sample loading and assay sensitivity .

How does cell cycle status affect RB1 Ser780 phosphorylation?

The phosphorylation of RB1 at Ser780 is tightly regulated throughout the cell cycle:

Cell cycle phase correlation:

• G0/G1 (quiescent/early G1): Minimal Ser780 phosphorylation

• Late G1: Increasing phosphorylation as cells approach G1/S transition

• S phase: High levels of Ser780 phosphorylation

• G2/M: Maintained high phosphorylation levels

Regulation mechanism:

• Cyclin D-CDK4/6 complexes are primarily responsible for phosphorylating RB1 at Ser780

• Cyclin D1 is specifically required for Ser780 phosphorylation in vivo

• Phosphorylation at Ser780 reduces RB1's binding to E2F transcription factors

Experimental manipulation:

• Serum starvation reduces phosphorylation (G0/G1 arrest)

• Nocodazole treatment (0.2 μg/mL) increases phosphorylation (G2/M arrest)

• CDK4/6 inhibitors (e.g., palbociclib) specifically reduce Ser780 phosphorylation

• Tetrandrine treatment (30 μM) reduces phosphorylation

When interpreting phosphorylation patterns, consider the cell cycle distribution of your sample population, as heterogeneous cultures will show variable phosphorylation levels. Flow cytometry can be particularly valuable for correlating phosphorylation status with cell cycle phase at the single-cell level .

How can phosphorylation at different RB1 sites be distinguished experimentally?

RB1 contains multiple phosphorylation sites with distinct functional implications. Distinguishing them requires specialized approaches:

Multiplex phosphorylation analysis:

• Use phospho-specific antibodies targeting different sites (e.g., Ser780, Ser807/811) in parallel experiments

• Employ multiplex Western blotting with different fluorophore-conjugated secondary antibodies

• Consider MSD MULTI-SPOT assays that can detect multiple phosphorylation sites simultaneously

Phosphorylation site-specific effects:

• Ser780 phosphorylation is specifically mediated by cyclin D-CDK4/6 complexes

• Ser807/811 can be phosphorylated by multiple CDKs including cyclin E-CDK2

• Different phosphorylation patterns correlate with distinct cellular contexts and functions

Advanced analytical approaches:

• Mass spectrometry-based phosphoproteomics for comprehensive site mapping

• Phospho-peptide enrichment using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC)

• Proximity ligation assays (PLA) to detect specific phosphorylated forms in situ

• FRET-based biosensors to monitor phosphorylation dynamics in living cells

When designing experiments to distinguish phosphorylation sites, consider using CDK-specific inhibitors to establish site-specific kinase relationships and lambda phosphatase treatment as a negative control for all phosphorylation events .

What methods are recommended for quantifying RB1 Ser780 phosphorylation in complex samples?

Accurate quantification of RB1 Ser780 phosphorylation requires careful methodological considerations:

Quantitative Western blotting:

• Always normalize phospho-signal to total RB1 protein levels

• Use fluorescent secondary antibodies for wider linear dynamic range

• Include a titration curve of control samples to ensure measurements fall within linear range

• Employ image analysis software with background subtraction capabilities

ELISA and MSD electrochemiluminescence assays:

• MSD MULTI-ARRAY assays provide sensitive quantification with better dynamic range than traditional ELISA

• Sample data from MSD assays shows clear distinction between phosphorylated and non-phosphorylated states:

Flow cytometry quantification:

• Enables correlation of phosphorylation with cell cycle phases

• Allows measurement at single-cell resolution

• Recommended antibody amount: 0.25 μg per 10^6 cells

• Requires careful fixation and permeabilization protocols

Absolute quantification approaches:

• Isotope-labeled peptide standards for mass spectrometry

• Recombinant phosphorylated protein standards for immunoassays

• Digital ELISA platforms for single-molecule detection sensitivity

For complex tissue samples, consider combining phospho-specific IHC with digital image analysis for spatial quantification or laser capture microdissection followed by immunoassays for region-specific measurements .

How can researchers troubleshoot inconsistent Phospho-RB1 (Ser780) antibody results?

When facing inconsistent results, consider these systematic troubleshooting approaches:

Sample preparation issues:

• Inadequate phosphatase inhibition leading to signal loss

  • Solution: Use fresh, complete inhibitor cocktails; keep samples cold

• Protein degradation affecting quantification

  • Solution: Add protease inhibitors; verify total RB1 signal integrity

• Incomplete denaturation for Western blotting

  • Solution: Ensure adequate heating in SDS sample buffer (95°C, 5 minutes)

Antibody-specific considerations:

• Batch-to-batch variability

  • Solution: Validate each new lot against previous standards

• Non-specific binding

  • Solution: Optimize blocking conditions; consider using 5% BSA instead of milk

• Sub-optimal antibody concentration

  • Solution: Titrate antibody across recommended range (e.g., 1:500-1:2000 for WB)

Technical variations:

• Inconsistent transfer efficiency in Western blotting

  • Solution: Use stain-free gels or total protein normalization

• Cell cycle heterogeneity affecting phosphorylation patterns

  • Solution: Synchronize cells or use flow cytometry for single-cell analysis

• Fixation artifacts in IHC/ICC

  • Solution: Optimize fixation protocol; test multiple fixatives

Validation approaches:

• Run known positive and negative controls in parallel

• Include phosphatase-treated samples as definitive negative controls

• Compare results across multiple detection methods (e.g., WB vs. ELISA)

• Verify results with alternative antibody clones targeting the same site

Document all experimental conditions thoroughly to identify variables that may contribute to inconsistency, including cell density, passage number, treatment duration, and lysis conditions .

How do RB1 mutations affect detection by Phospho-RB1 (Ser780) antibodies?

The impact of RB1 mutations on antibody detection requires careful consideration:

Mutation effects on epitope recognition:

• Missense mutations near Ser780 may alter antibody binding affinity

• Deletion mutations may remove the Ser780 site entirely

• Frameshift mutations can create premature stop codons (as in GOS561/GOS563 mutations) , potentially eliminating the phosphorylation site

Potential challenges with cancer cell lines:

• Many cancer cell lines harbor RB1 mutations or deletions

• Some cell lines express truncated RB1 proteins like p70-RB (C-terminal truncation)

• RB1-null cell lines won't produce signal regardless of antibody quality

Experimental approaches:

• Sequence verification of RB1 in your experimental system

• Western blotting with antibodies targeting different RB1 regions

• Co-detection with total RB1 antibody to verify protein expression

• Detection of alternative pocket proteins (p107/p130) that may compensate for RB1 loss

Cell line considerations:

• Known RB1 wild-type lines: MCF7, A549, HCT116

• Common RB1-mutant/null lines: SAOS-2, MDA-MB-468, NCI-H596

• When working with patient-derived samples, particularly from retinoblastoma, osteosarcoma, or small cell lung cancer, consider genetic testing for RB1 status

Understanding the genetic status of RB1 in your experimental system is crucial for accurate interpretation of phosphorylation data and can help explain unexpected negative results.

What are the latest methodologies for studying dynamic RB1 phosphorylation in living systems?

Recent advances have expanded the toolbox for studying RB1 phosphorylation dynamics:

Live-cell imaging approaches:

• FRET-based biosensors to monitor RB1 phosphorylation in real-time

• Fluorescently-tagged RB1 to track localization changes upon phosphorylation

• Optogenetic tools to induce rapid CDK activation and study phosphorylation kinetics

Single-cell analysis technologies:

• Mass cytometry (CyTOF) for high-dimensional analysis of RB1 phosphorylation alongside other markers

• Single-cell Western blotting for heterogeneity assessment

• Imaging flow cytometry combining visual and quantitative phosphorylation data

Pharmacological probing strategies:

• Selective CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) to specifically modulate Ser780 phosphorylation

• Rapid phosphatase activation systems to study dephosphorylation dynamics

• Degrader technologies (PROTACs) for acute depletion of relevant kinases

In vivo monitoring approaches:

• Patient-derived xenografts with sequential sampling to monitor treatment effects

• Phospho-proteomic analysis of tumor biopsies before and after treatment

• Immunohistochemistry panels incorporating phospho-RB1 (Ser780) with proliferation markers

Emerging technologies:

• CRISPR-Cas9 knock-in of tagged RB1 at endogenous loci

• Nanobody-based detection of phosphorylated RB1 in living cells

• Computational modeling of RB1 phosphorylation networks integrated with experimental data