ALPK1 antibody (N-Term)

Catalog No. ABIN3187581

This anti-ALPK1 antibody is a Rabbit Polyclonal antibody detecting ALPK1 in WB, ELISA, IHC and IF. Suitable for Human and Mouse. Independently validated for use in Western Blotting.

Quick Overview for ALPK1 antibody (N-Term) (ABIN3187581)

Target: ALPK1 (alpha-Kinase 1 (ALPK1))

Reactivity: Human, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This ALPK1 antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunohistochemistry (IHC), Immunofluorescence (IF)

Product Details anti-ALPK1 Antibody

Binding Specificity: N-Term

Specificity: α-protein Kinase 1 Polyclonal Antibody detects endogenous levels of α-protein Kinase 1 protein.

Characteristics: Rabbit Polyclonal to α-protein Kinase 1.

Purification: The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen.

Immunogen: Synthesized peptide derived from the N-terminal region of human alpha-protein Kinase 1.

Isotype: IgG

Application Details

Application Notes: WB 1:500-1:2000, IHC 1:100-1:300, IF 1:200-1:1000, ELISA 1:20000,

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103932 (Western Blotting)

by: Institute of Experimental Internal Medicine, Otto von Guericke Universität Magdeburg

No.: #103932

Date: 08/26/2019

Antigen: ALPK1

Lot Number: 63660

Method validated: Western Blotting

Positive Control:

Recombinant c-Myc-tagged hALPK1 overexpression in AGS KO^hALPK1

Negative Control:

AGS KO^hALPK1

Notes:

ABIN3187581 detects overexpressed hALPK1 but not the endogenous protein in AGS cells.

Validation Images

Full Methods

Primary Antibody: ABIN3187581

Secondary Antibody: donkey anti-rabbit HRP antibody (Jackson ImmunResearch, 711-036-152, lot 140655)

Full Protocol:

• Grow AGS (ATCC, CRL-1739 ) in RPMI 1640 (Gibco) supplemented with 10% FCS (Gibco) and no antibiotics, at 37°C and 5% CO₂ in 4ml on a 60mm dish to 70% confluency.
• Transfect cells with 5µg phALPK1 (Origene) using Metafectene Pro (Biontex) following the manufacturer´s instructions.
• Lyse 1.6x10⁶ cells in 70µl per dish of ice-cold M-PER (Pierce) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, 78440) after two PBS washes.
• Determine total protein content of the lysates using BCA (ThermoFisher).
• Denature 200µg of total protein for 5min at 95°C in 100µl and subsequently separate 20µg or 40µg on a freshly cast denaturing 10% polyacrylamide gel in a Bio-Rad Protean 3 for 15min at 100V and 1.5h at 140V.
• Transfer proteins onto membrane 0.45µm PVDF (Millipore, IPVH00010) with a Bio-Rad Western blotting system for 2h at 100V constant.
• Block the membrane with TBS-T and 5% skim milk (Roth) for 1 at RT.
• Incubation with primary rabbit-anti ALPK1 antibody (antibodies-online, ABIN3187581, lot 63660) diluted 1:1000 in TBS-T containing BSA (AppliChem, A1391, 0500) ON at 4°C.
• Wash membrane 3x for 5, 10, 10 minutes with TBS-T.
• Incubation with secondary donkey anti-rabbit HRP antibody (Jackson ImmunResearch, 711-036-152, lot 140655) diluted 1:10000 in TBS-T and 5% skim milk (Roth) for 1h at RT.
• Wash membrane 3x for 10min with TBS-T.
• Reveal protein bands using Immobilon Western (Millipore, WBKLS0500, lot 1827003) using ChemoCAM (INTAS) as gel documentation system.

Experimental Notes:

• ABIN3187581 detected very strong two unspecific bands running at approximately 60kDa and 95kDa. This was visible incubating the antibody in milk or albumin. In neither milk nor albumin the antibody was able to detect the endogenous hALPK1 present in AGS cells. Incubation in albumin but not milk lead to the detection of recombinant overexpressed hALPK1 as confirmed by c-Myc detection (SCBT, sc-40) of the recombinant protein. Another antibody available in our laboratory (Proteintech Europe 19107-1-AP, lot 00010221) also detects the overexpressed hALPK1 (~135kDa) but not the endogenous hALPK1.

• It is possible that the amount of ALPK1 in our cell preparation is low. Other cell lysate might be better suited. From my data, I can conclude that this antibody detects overexpressed hALPK1.

• Because no antibody detecting the endogenous hALPK1 is available to me, I cannot confirm the knockout in our cell lines, but on the functional level, we have indications for a successful knockout.

Handling

Format: Liquid

Concentration: 1 mg/mL

Buffer: Liquid in PBS containing 50 % glycerol, 0.5 % BSA and 0.02 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freeze/thaw cycles.

Storage: -20 °C

Storage Comment: Store at -20°C.

Target Details for ALPK1

Target: ALPK1 (alpha-Kinase 1 (ALPK1))

Alternative Name: alpha-Protein Kinase 1

Molecular Weight: 139 kDa

Gene ID: 80216

UniProt: Q96QP1