Antibodies, Proteins, Kits and Reagents for Life Science

274 732 ₸ Product size

100 µl 274 732 ₸

Order now and get it on Wednesday March 10, 2021

Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

Key features and details

Rabbit monoclonal [EP821Y] to Rb (phospho T373)
Suitable for: WB, IP, IHC-P, ICC/IF, Dot blot
Reacts with: Human
Isotype: IgG

Product name

Anti-Rb (phospho T373) antibody [EP821Y]
See all Rb primary antibodies
Description

Rabbit monoclonal [EP821Y] to Rb (phospho T373)
Host species

Rabbit
Specificity

ab52975 only detects Rb phosphorylated on Threonine 373.

Tested Applications & Species

Application	Species
ICC/IF	Human
IHC-P	Human
IP	Human
WB	Human

See all applications and species data

Immunogen

A synthetic phospho-peptide corresponding to residues surrounding Thr373 of human Rb was used as immunogen. The antibody only detects Rb phosphorylated on Threonine 373.
Positive control
- Jurkat nuclear cell lysate Human kidney carcinoma tissue
General notes

Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

Learn more here.

Form

Liquid
Storage instructions

Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage buffer

pH: 7.20
Preservative: 0.05% Sodium azide
Constituents: 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant
Concentration information loading...
Purity

Tissue culture supernatant
Clonality

Monoclonal
Clone number

EP821Y
Isotype

IgG
Research areas

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

ab52975, at a 1/250 dilution, staining Human Rb (Phosphorylated) in Kidney carcinoma, using Immunohistochemistry, Paraffin Embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/10000 dilution

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) serum starved for 24 h. Then 10% FBS incubated for 20 h. Whole cell lysates.
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) serum starved for 24 h. Then 10% FBS incubated for 20 h.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 106 kDa
Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.

Exposure time: 1 minute

Blocking and diluting buffer: 5% NFDM/TBST

The different result is due to the lysates preparation method. The lysate in lane 1 is prepared by 1%SDS Hot Lysate buffer method. The lysate in lane 2 is prepared by RIPA lysis method. This antibody works in 1%SDS Hot Lysates in WB.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
Immunocytochemistry/ Immunofluorescence - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized MCF7(Human breast denocarcinoma cell line) cells, Serum starved and non-starved, labeling Rb (phospho T373) with Ab52975 at 1/100 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor^®488 (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on MCF7 cells.The number of positive cells increased after treatment with FBS (fetal bovine serum) for 48 hours, then decreased after Lambda Protein Phosphatase treatment (31℃ for 2hours ).
Immunoprecipitation - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/500 dilution

Lane 1 : Jurkat (human acute T cell leukemia) starved 24h then 10% FBS incubated for 20h whole cell lysate.
Lane 2 : Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52975 in Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate.

Lysates/proteins at 10 µg per lane.

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size : 106 kDa
Additional bands at : 110 kDa.

Exposure time : 1 second
Blocking and diluting buffer: 5% NFDM/TBST

The IP lysates are prepared by RIPA method. As the WB image shows, this antibody works in 1%SDS Hot Lysate buffer in WB. So there is no band in input lane.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

Lanes 1-3 : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/500 dilution

Lane 1 : Jurkat (human acute T cell leukemia) starved 24h then 10% FBS incubated for 20h whole cell lysate. at 10 µg
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab52975 in Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate. at 10 µg

Secondary
Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.

Exposure time: 1 second
Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/10000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) 2 day serum starved whole cell lysate.
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate.
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate. Membrane incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 106 kDa
Observed band size: 110 kDa
why is the actual band size different from the predicted?

Exposure time: 30 seconds

The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
Dot Blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

Dot Blot analysis of Lane 1: Rb (pT373) phospho peptide and Lane 2: Rb non-phospho peptide labeling Rb (phospho T373) with ab52975 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.
Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/50000 dilution

Lane 1 : Jurkat cell lysate (untreated)
Lane 2 : Jurkat cell lysate (treated with FBS)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : goat anti-rabbit HRP labelled at 1/2000 dilution

Predicted band size: 106 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

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