RNF40 Antibody (Rabbit mAb) [A17P16]

Application: Reactivity: Mouse, Rat, Human

Lane 1: HeLa, Lane 2: C6, Lane 3: RAW 264.7, Lane 4: PC-12

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:80 IHC1:100

Application
WB, IP, IHC

Reactivity
Mouse, Rat, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
114 kDa 130 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human fetal liver tissue; Human fetal heart tissue; Human fetal kidney tissue; Human colon tissue; Mouse colon tissue; Rat colon tissue; HeLa cells; HEK-293 cells; mESC cells; MCF7 cells; A-375 cells; HepG2 cells; C6 cells; RAW 264.7 cells; PC-12 cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
RNF40 Antibody (Rabbit mAb) [A17P16] detects endogenous levels of total RNF40 protein.

Subcellular Location
Nucleus

Uniprot ID
O75150

Clone
A17P16

Synonym(s)
BRE1B, KIAA0661, RNF40, E3 ubiquitin-protein ligase BRE1B, BRE1-B, 95 kDa retinoblastoma-associated protein, RING finger protein 40, RING-type E3 ubiquitin transferase BRE1B, RBP95

Background
RNF40 is a RING‑finger E3 ubiquitin ligase that forms a stable heterodimer with its paralog RNF20 to generate the principal mammalian complex responsible for monoubiquitination of histone H2B at lysine 120, a modification that shapes the histone code and controls transcriptional programs in a context‑dependent manner by influencing downstream histone H3K4 and H3K79 methylation, transcriptional elongation and enhancer activity. The RNF20/40 complex contains multiple coiled‑coil regions that mediate heterodimerization and interaction with chromatin‑associated factors, and a C‑terminal RING domain on RNF40 that engages E2 ubiquitin‑conjugating enzymes such as UBE2A/UBE2B to catalyze H2B monoubiquitination at actively transcribed regions; conditional deletion of Rnf40 in mouse embryonic fibroblasts selectively affects genes marked by low to moderate H2Bub1, with decreased expression of these RNF40‑dependent genes correlating with narrowing of H3K4me3 peaks and reduced transcriptional elongation, while genes with high H2Bub1 levels remain largely unchanged. RNF40 loss also leads to upregulation of a subset of genes enriched for H3K27me3 due to decreased expression of the Ezh2 methyltransferase, and this epigenetic shift is accompanied by enhancer activation through FOXL2 at some loci, showing that RNF40‑controlled H2Bub1 can either promote or restrain transcription depending on the surrounding chromatin landscape and the interplay with other histone marks and regulators. In the context of viral infection by Kaposi’s sarcoma‑associated herpesvirus (KSHV), RNF40 and RNF20 are directly utilized by the viral immediate‑early transcription factor RTA/ORF50: proteomics analysis identifies a specific interaction between RTA and the RNF20/40 complex, and genetic or pharmacologic interference with RNF20/40 activity reduces RTA‑induced lytic reactivation, lowers expression of lytic genes including RTA itself and other replication factors, and diminishes production of infectious virions, indicating that RTA co‑opts the host H2B ubiquitin ligase to remodel chromatin at lytic promoters and drive the switch from latency to the lytic cycle. RNF40 thus connects a core epigenetic modification—H2BK120ub1—with both host gene regulation and viral life‑cycle control, participating in chromatin‑based regulation of developmental and tissue‑specific transcription under basal conditions and being recruited by KSHV RTA to enforce viral lytic gene expression in infected B cells, a mechanism that supports viral dissemination and contributes to the pathogenesis of KSHV‑associated malignancies.

Background

RNF40 is a RING‑finger E3 ubiquitin ligase that forms a stable heterodimer with its paralog RNF20 to generate the principal mammalian complex responsible for monoubiquitination of histone H2B at lysine 120, a modification that shapes the histone code and controls transcriptional programs in a context‑dependent manner by influencing downstream histone H3K4 and H3K79 methylation, transcriptional elongation and enhancer activity. The RNF20/40 complex contains multiple coiled‑coil regions that mediate heterodimerization and interaction with chromatin‑associated factors, and a C‑terminal RING domain on RNF40 that engages E2 ubiquitin‑conjugating enzymes such as UBE2A/UBE2B to catalyze H2B monoubiquitination at actively transcribed regions; conditional deletion of Rnf40 in mouse embryonic fibroblasts selectively affects genes marked by low to moderate H2Bub1, with decreased expression of these RNF40‑dependent genes correlating with narrowing of H3K4me3 peaks and reduced transcriptional elongation, while genes with high H2Bub1 levels remain largely unchanged. RNF40 loss also leads to upregulation of a subset of genes enriched for H3K27me3 due to decreased expression of the Ezh2 methyltransferase, and this epigenetic shift is accompanied by enhancer activation through FOXL2 at some loci, showing that RNF40‑controlled H2Bub1 can either promote or restrain transcription depending on the surrounding chromatin landscape and the interplay with other histone marks and regulators. In the context of viral infection by Kaposi’s sarcoma‑associated herpesvirus (KSHV), RNF40 and RNF20 are directly utilized by the viral immediate‑early transcription factor RTA/ORF50: proteomics analysis identifies a specific interaction between RTA and the RNF20/40 complex, and genetic or pharmacologic interference with RNF20/40 activity reduces RTA‑induced lytic reactivation, lowers expression of lytic genes including RTA itself and other replication factors, and diminishes production of infectious virions, indicating that RTA co‑opts the host H2B ubiquitin ligase to remodel chromatin at lytic promoters and drive the switch from latency to the lytic cycle. RNF40 thus connects a core epigenetic modification—H2BK120ub1—with both host gene regulation and viral life‑cycle control, participating in chromatin‑based regulation of developmental and tissue‑specific transcription under basal conditions and being recruited by KSHV RTA to enforce viral lytic gene expression in infected B cells, a mechanism that supports viral dissemination and contributes to the pathogenesis of KSHV‑associated malignancies.

References
https://pubmed.ncbi.nlm.nih.gov/28209164/ https://pubmed.ncbi.nlm.nih.gov/37888983/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%