Anti-Rb antibody [EPR17512]

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Western blot : Anti-RB1 antibody [EPR17512] (ab181616) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab181616 was shown to bind specifically to RB1. A band was observed at 106 kDa in wild-type A549 cell lysates with no signal observed at this size in RB1 knockout cell line. To generate this image, wild-type and RB1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 40 µg

Lanes 2 - 3:

RB1 knockout A549 cell lysate at 40 µg

Lanes 4 - 5:

HeLa cell lysate at 20 µg

Lane 6:

Wild-type HAP1 cell lysate at 20 µg

Lane 7:

RB1 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 106 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Rb antibody [EPR17512] (AB181616)

ab181616 staining Rbin the human cell line MCF-7 (human breast carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/140. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Rb with ab181616 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab181616 anti-Rb antibody [EPR17512] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on cancer cells of Human breast cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human lung is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Rb antibody [EPR17512] (AB181616)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling Rb with ab181616 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on MCF7 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab181616 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Rb with ab181616 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab181616 anti-Rb antibody [EPR17512] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IP

Supplier Data

Immunoprecipitation - Anti-Rb antibody [EPR17512] (AB181616)

Rb was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab181616 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab181616 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : MCF7 whole cell lysate 10 μg (Input). Lane 2 : ab181616 IP in MCF7 whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab181616 in MCF7 whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Rb antibody [EPR17512] (ab181616)

Predicted band size: 106 kDa

false

Exposure time: 8s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse lung is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616)

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on neuron of mouse cerebral cortex is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Palbociclib is a CDK4/6 inhibitor. Treatment leads to a reduction in total Rb1 levels through G1 phase arrest. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (<a href='/en-us/products/primary-antibodies/rb-phospho-t252-t356-s608-s780-s807-antibody-rm1144-ab320747'>ab320747</a>) at 1/1000 dilution

Lane 1:

Untreated Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Jurkat treated with 1uM palbociclib for 48 hours whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated Jurkat whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 4:

Jurkat treated with 1uM palbociclib for 8 hours whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Rb knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HEK293 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab181616 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181616 was shown to recognize Rb in wild-type HAP1 cells as signal was lost at the expected MW in Rb knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and RB1 knockout samples were subjected to SDS-PAGE. ab181616 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616)

Predicted band size: 106 kDa

false

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

This blot was developed using a high sensitivity ECL substrate.

The identity of the bands at approximately 40 kDa, 60 kDa and 280 kDa (in lanes 1-2) are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (<a href='/en-us/products/primary-antibodies/rb-phospho-s795-antibody-epr28156-6-ab321974'>ab321974</a>) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) serum starvation for 48h, whole cell lysate (untreated membrane) at 50 µg

Lane 2:

MCF7 serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (untreated membrane) at 50 µg

Lane 3:

MCF7 serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (alkaline phosphatase treated membrane) at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 106 kDa,36 kDa

true

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, ab320747 was shown to bind specifically to RB1. Target of interest was observed at 100 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in RB1 knockout cell line (lane 2). In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (<a href='/en-us/products/primary-antibodies/rb-phospho-t252-t356-s608-s780-s807-antibody-rm1144-ab320747'>ab320747</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

RB1 knockout HAP1 whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Wild-type HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 4:

RB1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Western blot : Rabbit monoclonal [EPR17512] to Rb ab181616 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 106 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in RB1 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human RB1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-rb1-knockout-hct116-cell-line-ab286657'>ab286657</a>) at 20 µg

Lane 3:

Wild-type A549 ab288558 at 20 µg

Lane 4:

Western blot - Human RB1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rb1-knockout-a549-cell-line-ab286470'>ab286470</a>) at 20 µg

Lane 5:

HEK-293 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 106 kDa

Observed band size: 106 kDa,51 kDa

false

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking/Dilution buffer : 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/20000 dilution

Lane 1:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 20 µg

Lane 3:

WEHI-3 (Mouse leukemia) whole cell lysate at 20 µg

Lane 4:

COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate at 20 µg

Lane 5:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 106 kDa,42 kDa

Observed band size: 105 kDa,42 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking/Dilution buffer : 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/2000 dilution

All lanes:

Human fetal brain lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 106 kDa,188 kDa,82 kDa

Observed band size: 105 kDa,188 kDa,82 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time :

Lane 1 and 2 : 100 seconds
Lane 3 and 4 : 10 seconds

We recommend to use 1%SDS Hot lysis method to get clear band.
We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/1000 dilution

Lane 1:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method at 20 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method at 20 µg

Lane 3:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in RIPA lysis method at 20 µg

Lane 4:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

false

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 1% BSA/TBST.

Performed under reducing conditions.

In Western blot, ab323846 was shown to bind specifically to Rb. Target of interest was observed at 106 kDa in Calyculin A treated wild-type HAP1 cell lysates (lane 2) with no signal observed at this size in Calyculin A treated Rb knockout cell line (lane 4).

Lanes 1-4 on the left untreated membrane with alkaline phosphatase; Lanes 1-4 on the right treated membrane with alkaline phosphatase.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512] - (ab181616) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Rb (phospho T821 + T826) antibody [EPR28648-126] (<a href='/en-us/products/primary-antibodies/rb-phospho-t821-t826-antibody-epr28648-126-ab323846'>ab323846</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate (left: untreated membrane with alkaline phosphatase) at 20 µg

Lane 1:

Untreated Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate (right: treated membrane with alkaline phosphatase) at 20 µg

Lane 2:

Wild-type HAP1 treated with 100ng/ml Calyculin A for 30min whole cell lysate (left: untreated membrane with alkaline phosphatase) at 20 µg

Lane 2:

Wild-type HAP1 treated with 100ng/ml Calyculin A for 30min whole cell lysate (right: treated membrane with alkaline phosphatase) at 20 µg

Lane 3:

Untreated Rb knockout HAP1 whole cell lysate (left: untreated membrane with alkaline phosphatase) at 20 µg

Lane 3:

Untreated Rb knockout HAP1 whole cell lysate (right: treated membrane with alkaline phosphatase) at 20 µg

Lane 4:

Rb knockout HAP1 treated with 100ng/ml Calyculin A for 30min whole cell lysate (left: untreated membrane with alkaline phosphatase) at 20 µg

Lane 4:

Rb knockout HAP1 treated with 100ng/ml Calyculin A for 30min whole cell lysate (right: treated membrane with alkaline phosphatase) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 106 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (<a href='/en-us/products/primary-antibodies/rb-phospho-s795-antibody-epr28156-6-ab321974'>ab321974</a>) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) serum starvation for 48h, whole cell lysate (untreated membrane) at 50 µg

Lane 2:

Jurkat serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (untreated membrane) at 50 µg

Lane 3:

Jurkat serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (alkaline phosphatase treated membrane) at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 106 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking/Dilution buffer : 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/10000 dilution

Lane 1:

F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg

Lane 2:

Mouse brain lysate at 10 µg

Lane 3:

Mouse lung lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 106 kDa

Observed band size: 105 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (<a href='/en-us/products/primary-antibodies/rb-phospho-s795-antibody-epr28156-6-ab321974'>ab321974</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with a human Rb expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human Rb (S795A mutation) expression vector containing a His-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 106 kDa,124 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-Rb antibody [EPR17512] (AB181616)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (<a href='/en-us/products/primary-antibodies/rb-phospho-t252-t356-s608-s780-s807-antibody-rm1144-ab320747'>ab320747</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-his tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human Rb1 expression vector containing a myc-his-tag, whole cell lysate at 2 µg

Lane 3:

293T cells transfected with a human Rb1(T252A, T56A, S608A, S780A, S807A) expression vector containing a myc-his-tag, whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 15s