Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling PIM1 with ab322238 at 1/50 (10.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic and nuclear staining in K-562 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab322238 [EPR28184-93]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab322238 [EPR28184-93]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab322238 [EPR28184-93]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IP

Supplier Data

Immunoprecipitation - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

PIM1 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab322238 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322238 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : ab322238 IP in K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322238 in K-562 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-PIM1 antibody [EPR28184-93] (<a href='/en-us/products/primary-antibodies/pim1-antibody-epr28184-93-ab322238'>ab322238</a>) at 1/30 dilution

All lanes:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

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 1/5000 dilution

false

Exposure time: 58s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized F9 (mouse embryonal carcinoma epithelial cell) cells labelling PIM1 with ab322238 at 1/50 (10.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic and nuclear staining in F9 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

PIM1 was immunoprecipitated from 0.35 mg F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate with ab322238 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322238 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate
Lane 2 : ab322238 IP in F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322238 in F9 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

Larger 44 kDa (PIM1-L) is active kinase, but differ in stability (PMID : 1825810).

All lanes:

Immunoprecipitation - Anti-PIM1 antibody [EPR28184-93] (<a href='/en-us/products/primary-antibodies/pim1-antibody-epr28184-93-ab322238'>ab322238</a>) at 1/30 dilution

All lanes:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate

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 1/5000 dilution

false

Exposure time: 58s

• WB

Supplier Data

Western blot - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

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

The antibody detects both the larger 44 kDa isoform (Pim-1L) and the smaller 33 kDa isoform (Pim-1S). Both isoforms function as active kinases, but differ in stability (PMID : 1825810).

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

Exposure time : Lane 1-2 : 15 seconds; Lane 3-7 : 114 seconds

All lanes:

Western blot - Anti-PIM1 antibody [EPR28184-93] (<a href='/en-us/products/primary-antibodies/pim1-antibody-epr28184-93-ab322238'>ab322238</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) transfected with scrambled siRNA control whole cell lysate at 40 µg

Lane 2:

K-562 transfected with siRNA specifically targeting PIM1 whole cell lysate at 40 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

HDLM-2 (human Hodgkin lymphoma cell) whole cell lysate at 40 µg

Lane 5:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 6:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 40 µg

Lane 7:

4T1 (mouse mammary gland carcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 35 kDa,44 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-PIM1 antibody [EPR28184-93] - BSA and Azide free (AB324767)

This data was developed using ab322238, the same antibody clone in a different buffer formulation.

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

This antibody does not cross-react with PIM-3.

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

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

All lanes:

Western blot - Anti-PIM1 antibody [EPR28184-93] (<a href='/en-us/products/primary-antibodies/pim1-antibody-epr28184-93-ab322238'>ab322238</a>) at 1/1000 dilution

Lane 1:

His-tagged human PIM-1 recombinant fragment at 10 ng

Lane 2:

His-tagged human PIM-3 recombinant protein at 10 ng

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: 35 kDa,36 kDa

false

Exposure time: 180s