Anti-Rb (phospho T373) antibody [EP821Y] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Rb (phospho T373) antibody [EP821Y] - BSA and Azide free (AB219158)

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°C for 2hours ).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52975).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T373) antibody [EP821Y] - BSA and Azide free (AB219158)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52975).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-Rb (phospho T373) antibody [EP821Y] - BSA and Azide free (AB219158)

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 section of the website and/or here.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52975).

All lanes:

Immunoprecipitation - Anti-Rb (phospho T373) antibody [EP821Y] (<a href='/en-us/products/primary-antibodies/rb-phospho-t373-antibody-ep821y-ab52975'>ab52975</a>) 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 2:

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 (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/rb-phospho-t373-antibody-ep821y-ab52975'>ab52975</a> in Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate. at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 106 kDa

Observed band size: 110 kDa

false

Exposure time: 1s

• Dot

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Dot Blot - Anti-Rb (phospho T373) antibody [EP821Y] - BSA and Azide free (AB219158)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52975).