Recombinant Anti-PBK/SPK antibody [EPR21982] KO Tested (ab236871)

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Western blot - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR21982] to PBK/SPK
Suitable for: Flow Cyt (Intra), WB, IHC-P, IP
Knockout validated
Reacts with: Rat, Human

Carrier Free

Product name

Anti-PBK/SPK antibody [EPR21982]
See all PBK/SPK primary antibodies
Description

Rabbit monoclonal [EPR21982] to PBK/SPK
Host species

Rabbit
Specificity

IHC is recommended for human only.
Tested applications

Suitable for: Flow Cyt (Intra), WB, IHC-P, IPmore details
Species reactivity

Reacts with: Rat, Human
Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: Wild-type HAP1 cell lysate; HEK293T, HeLa, HepG2, A431, U-87 MG, C6 and PC-12 whole cell lysates; Rat testis lysate. IHC-P: Human testis, gastric cancer and esophagus tissues. Flow Cyt (intra): HeLa and HepG2 cells. IP: HeLa whole cell lysate.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR21982
Isotype

IgG
Research areas

Alternative Versions
- Anti-PBK/SPK antibody [EPR21982] - BSA and Azide free (ab239756)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human PBK (SPK) knockout HEK-293T cell line (ab266827)
KO cell lysates
- Human PBK (SPK) knockout HEK-293T cell lysate (ab257575)
Recombinant Protein
- Recombinant human PBK/SPK protein (ab85275)
Related Products
- Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) (ab93684)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab236871 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
Flow Cyt (Intra)		1/50.
WB		1/1000. Detects a band of approximately 38 kDa (predicted molecular weight: 36 kDa).
IHC-P		1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. IHC is recommended for human only.
IP		1/30.

Notes
Flow Cyt (Intra) 1/50.
WB 1/1000. Detects a band of approximately 38 kDa (predicted molecular weight: 36 kDa).
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. IHC is recommended for human only.
IP 1/30.

Function

Phosphorylates MAP kinase p38. Seems to be active only in mitosis. May also play a role in the activation of lymphoid cells. When phosphorylated, forms a complex with TP53, leading to TP53 destabilization and attenuation of G2/M checkpoint during doxorubicin-induced DNA damage.
Tissue specificity

Expressed in the testis and placenta. In the testis, restrictedly expressed in outer cell layer of seminiferous tubules.
Sequence similarities

Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. MAP kinase kinase subfamily.
Contains 1 protein kinase domain.
Post-translational
modifications

Phosphorylated; in a cell-cycle dependent manner at mitosis.
Target information above from: UniProt accession Q96KB5 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 55872 Human
- Entrez Gene: 290326 Rat
- Omim: 611210 Human
- SwissProt: Q96KB5 Human
- Unigene: 104741 Human
Alternative names
- Cancer/testis antigen 84 antibody
- CT84 antibody
- Epididymis luminal protein 164 antibody
- FLJ14385 antibody
- HEL164 antibody
- Lymphokine activated killer T cell originated protein kinase antibody
- Lymphokine-activated killer T-cell-originated protein kinase antibody
- MAPKK like protein kinase antibody
- MAPKK-like protein kinase antibody
- Nori 3 antibody
- Nori-3 antibody
- Nori3 antibody
- PBK antibody
- PDZ binding kinase antibody
- PDZ-binding kinase antibody
- Serine/threonine protein kinase antibody
- Spermatogenesis related protein kinase antibody
- Spermatogenesis-related protein kinase antibody
- SPK antibody
- T LAK cell originated protein kinase antibody
- T-LAK cell-originated protein kinase antibody
- TOPK antibody
- TOPK_HUMAN antibody
see all

Western blot - Anti-PBK/SPK antibody [EPR21982] (ab236871)

All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PBK knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab236871 observed at 40 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab236871 was shown to react with PBK/SPK in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266827 (knockout cell lysate ab257575) was used. Wild-type HEK-293T and PBK knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab236871 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in germ cells of human testis (PMID:16982762; PMID:25909225) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
Flow Cytometry (Intracellular) - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling PBK/SPK with ab236871 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.
Western blot - Anti-PBK/SPK antibody [EPR21982] (ab236871)

All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : PBK/SPK knockout HAP1 cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:23547718; PMID:25909225).

ab236871 was shown to specifically react with PBK/SPK in wild-type HAP1 cells as signal was lost in PBK/SPK knockout cells. Wild-type and PBK/SPK knockout samples were subjected to SDS-PAGE. ab236871 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.
Western blot - Anti-PBK/SPK antibody [EPR21982] (ab236871)

All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : SW480 (human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 2 : A431 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 3 : U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg
Lane 4 : Rat testis lysate at 20 µg
Lane 5 : C6 (rat glial tumor cell line) whole cell lysate at 10 µg
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Exposure time : Lanes 1-3: 26 seconds; Lane 4: 8 seconds; Lanes 5-6: 70 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:23547718; PMID:25909225).
Immunoprecipitation - Anti-PBK/SPK antibody [EPR21982] (ab236871)

PBK/SPK was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab236871 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab184276 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg (Input).

Lane 2: ab231871 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab231871 in HeLa whole cell lysate.

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

Exposure time: 30 seconds.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Immunohistochemical analysis of paraffin-embedded human esophagus cancer tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in human esophagus cancer (PMID: 27919968) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in human gastric cancer (PMID:26894977; PMID:27898655) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
Flow Cytometry (Intracellular) - Anti-PBK/SPK antibody [EPR21982] (ab236871)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cell line labeling PBK/SPK with ab236871 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.
Anti-PBK/SPK antibody [EPR21982] (ab236871)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

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Publishing research using ab236871? Please let us know so that we can cite the reference in this datasheet.

ab236871 has not yet been referenced specifically in any publications.

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

KO cell lines

KO cell lysates

Recombinant Protein

Related Products

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Post-translationalmodifications

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (0)

Customer reviews and Q&As

Post-translational
modifications