RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

Novus Biologicals | Catalog # NB200-598

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse, Primate (Negative), Yeast

Cited:

Human, Mouse, Yeast

Predicted:

Drosophila (100%), Hamster (100%), Plant (100%). Backed by our 100% Guarantee.

Applications

Validated:

Western Blot, ELISA, Flow Cytometry, Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), CyTOF-ready

Cited:

Western Blot, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), Chemotaxis

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # 4H8

Format

Azide and BSA Free
View all RNA Polymerase II/POLR2A Primary Antibodies »

Product Specifications

Immunogen

10 repeats of synthetic peptide YSPTSPS using chemically synthesized phospho-Ser 5 YSPTSpPS (Human). [UniProt# P24928]

Reactivity Notes

Does not cross react with monkey.

Specificity

Please note that this antibody detects both unphosphorylated and phosphorylated forms of the protein. When using this antibody in western blot and IP, it recognizes both the II0 and IIA form of RPB1 (ie the largest RNAPII subunit). These different forms of RPB1 (as seen in gels) have traditionally been loosely defined as 'hyper-' and 'hypo-phosphorylated', respectively. Here, however, it is important to remember that RPB1 contains no less than 52 repeats of a heptapeptide (TSPTSPS) that can be phosphorylated at different positions (Tyr1, Ser2, Thr4, Ser5 and Ser7; this antibody was raised against a synthetic peptide with 10 of these repeats, mostly but probably not entirely, chemically phosphorylated at Ser5). So, when we say that it recognizes both forms (II0/hyper-phos and IIA/hypo-phos), it might really just mean that the antibody recognizes a few phosphorylated Ser5s among 52 otherwise mostly un-phosphorylated repeats. Alternatively, it might conceivably (though less likely) recognize UN-phosphorylated repeats as there might conceivably be a few of those even in the II0/hyper-phos RPB1 form.'

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Theoretical MW

217 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

Western Blot: RNA Polymerase II/POLR2A Antibody (4H8)Azide and BSA Free [NB200-598]

Western Blot: RNA Polymerase II/POLR2A Antibody (4H8)Azide and BSA Free [NB200-598]

Western Blot: RNA Polymerase II/POLR2A Antibody (4H8) [NB200-598] - Whole cell protein from HeLa (lane 1), MCF7 (lane 2), NIH-3T3 (lane 3) and Cos7 (lane 4) were separated by SDS-PAGE on a 6% gel, transferred to PVDF and probed with 2 ug/mL NB200-598. RNA Pol II reactivity (arrowhead) is observed in HeLa, MCF and 3T3 cells.
Immunocytochemistry/ Immunofluorescence: RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Immunocytochemistry/ Immunofluorescence: RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Immunocytochemistry/Immunofluorescence: RNA Polymerase II/POLR2A Antibody (4H8) [NB200-598] - RNA Polymerase II/POLR2A antibody was tested in HeLa cells with DyLight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and DyLight 550 (red).
Flow Cytometry: RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Flow Cytometry: RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Flow Cytometry: RNA Polymerase II/POLR2A Antibody (4H8) [NB200-598] - An intracellular stain was performed on HeLa cells with RNA Polymerase II/POLR2A (4H8) antibody NB200-598PE (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes. Both antibodies were conjugated to phycoerythrin.
Western Blot: RNA Polymerase II/POLR2A Antibody (4H8)Azide and BSA Free [NB200-598]

Western Blot: RNA Polymerase II/POLR2A Antibody (4H8)Azide and BSA Free [NB200-598]

Western Blot: RNA Polymerase II/POLR2A Antibody (4H8) [NB200-598] - Analysis of MCF7 cell lysata (A) using RNA polymerase II antibody at 2 ug/mL.
Flow (Intracellular): RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Flow (Intracellular): RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free [NB200-598]

Flow (Intracellular): RNA Polymerase II/POLR2A Antibody (4H8) [NB200-598] - An intracellular stain was performed on Daudi cells with RNA Polymerase II/POLR2A (4H8) antibody NB200-598 (blue) and a matched isotype control NBP2-27287 (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes, followed by mouse F(ab)2 IgG (H+L) APC-conjugated secondary antibody (F0101B, R&D Systems).

Applications for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

Application
Recommended Usage

Chromatin Immunoprecipitation (ChIP)

1:10-1:500

ELISA

1:100-1:2000

Flow Cytometry

1 ug per million cells

Immunocytochemistry/ Immunofluorescence

1:100-1:1000

Immunoprecipitation

1:10-1:500

Western Blot

1-2 ug/ml
Application Notes
In WB a band can be seen at approximately 217 kDa. Optimal dilutions/concentrations should be determined by the end user.

Reviewed Applications

Read 1 review rated 5 using NB200-598 in the following applications:

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

Azide and BSA Free

Preservative

No Preservative

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: RNA Polymerase II/POLR2A

RNA polymerase II (RNAPII) is a 12-subunit enzyme which synthesizes all mRNAs as well as several non-coding RNAs in eukaryotic cells thereby playing a central role in cellular functions. RNAPII is a DNA-dependent RNA polymerase enzyme which catalyzes the transcription of DNA into RNA employing four ribonucleoside triphosphates as substrates. The activity of RNAPII transcription machinery is regulated by DNA-binding transcriptional regulators in a gene/cell type-specific manner, whereas general transcription factors act as RNAPII accessory proteins required for the transcription of most class II genes, and the co-activators/co-repressors serve as bridges between DNA-bound factors and the RNAPII machinery, some affecting the organization and/or chemical modification of the chromatin template of RNAPII. RNA polymerase II subunit B1 (POLR2A) is the largest and the catalytic component of RNAPII which forms the polymerase active center together with the second largest subunit and is the central component of the basal RNAPII transcription machinery. It is composed of mobile elements that move relative to each other and POLR2A is the part of the core element with the central large cleft, the clamp element that moves to open and close the cleft and the jaws that are thought to grab the incoming DNA template.

Long Name

DNA-directed RNA Polymerase II Subunit RPB1

Alternate Names

POLR2, POLR2A, POLRA, RPB1, RPO2, RPOL2

Entrez Gene IDs

5430 (Human)

Gene Symbol

POLR2A

UniProt

P24928 (Human)

Additional RNA Polymerase II/POLR2A Products

Product Documents for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

Certificate of Analysis

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Product Specific Notices for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Related Research Areas

Epigenetics

Citations for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

Customer Reviews for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free (1)

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  • Name: Bryan Tinsley
    Application: Western Blot
    Sample Tested:
    Species: Human
    Verified Customer | Posted 05/13/2014
    Substrate of fourteen repeats of YSPTSPS fused to GST for purification (~50kD) from bacteria.
    RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free NB200-598

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Protocols

View specific protocols for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free (NB200-598):

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.

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FAQs for RNA Polymerase II/POLR2A Antibody (4H8) - Azide and BSA Free

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  • Q: The immunogen info mentions 10 repeats of synthetic peptide YSPTSPS. What does this mean?

    A: It is correct that the immunogen is literally 10 adjacent repeats of the sequence YSPTSPS, where the Ser 5 of each repeat is phosphorylated (the 'p Ser5' in the product name referring to serine 5 of this repeat rather than of the whole target protein). The datasheet for NB200-598 refers to UniProt #P24928. If you look at this sequence, you will see that it does contain these repeats of YSPTSPS; I ran a sequence comparison of this protein and 10x YSPTSPS and it aligned perfectly. For further information, the 'Background' section at the bottom of the datasheet for NB200-598 says, 'Recruitment of RNA processing factors to the elongation complex is coordinated by the phosphorylation of Serine-2 and Serine-5 residues within the C-terminal repeat (YSPTSPS) domain of the largest subunit of RNAPII'; and the 'Specificity' section near the top says, 'This antibody recognizes the C-terminal repeat of the largest subunit of RNA polymerase II. It recognizes both unphosphorylated and phosphorylated forms'.

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