GRK6 antibody (C-Term)

Catalog No. ABIN3042435

Cited in 1 publication. The Rabbit Polyclonal anti-GRK6 antibody (ABIN3042435) specifically detects GRK6 in WB. The antibody is reactive with Human samples. Independently validated for use in Western Blotting.

Quick Overview for GRK6 antibody (C-Term) (ABIN3042435)

Target: GRK6 (G Protein-Coupled Receptor Kinase 6 (GRK6))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This GRK6 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-GRK6 Antibody

Binding Specificity: C-Term

Purpose: Anti-GRK6 Antibody

Sequence: QSPFQQRKKK IKREEVERLV KEVPEEYSER FSPQAR

Specificity: No cross reactivity with other proteins.

Cross-Reactivity (Details): No cross-reactivity with other proteins

Predicted Reactivity: different from the related mouse sequence by four amino acids,and from the related rat sequence by three amino acids.

Characteristics: Anti-GRK6 Antibody. Tested in WB applications. This antibody reacts with Human.

Purification: Immunogen affinity purified.

Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human GRK6, different from the related mouse sequence by four amino acids, and from the related rat sequence by three amino acids.

Isotype: IgG

Application Details

Application Notes: Western blot, 0.1-0.5 μg/mL, Human

Comment:

Antibody can be supported by chemiluminescence kit ABIN921124 in WB.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103590 (Western Blotting)

by: Institut für Molekulare Zellbiologie, Universitätsklinikum Jena

No.: #103590

Date: 01/31/2019

Antigen: GRK6

Lot Number: 0971512Da9609109

Method validated: Western Blotting

Positive Control:

HEK293 cells transiently transfected with pcDNA3 vectors containing GRK6 transcript variant 1 (NM_001004106.3), transcript variant 2 (NM_002082.3), transcript variant 3 (NM_001004105.2), or transcript variant 4 (NM_001364164.1)

Negative Control:

HEK293 cells transiently transfected with empty pcDNA3 vector

Notes:

The GRK6 antibody ABIN3042435 is able to detect all four tested GRK6 isoforms, but gives strong background-signals around 55kDa.

Validation Images

Western blot analysis of HEK293 cell lysates subsequently to transient transfection with pcDNA3 constructs containing no insert (EV), GRK6 transcript variant 1, NM_001004106.3, transcript variant 2, NM_002082.3, transcript variant 3, NM_001004105.2, or transcript variant 4, NM_001364164.1 as indicated. Equal amounts of the lysates were loaded twice on the same gel and after blotting, the membrane was cut and incubated with ABIN3042435, a reference anti-GRK6 antibody (GRK6), and Vinculin loading control antibody (Vinculin) in parallel. Exposure for the upper panel was 15 seconds and for the lower 90 seconds.

Full Methods

Primary Antibody: ABIN3042435

Secondary Antibody: goat-anti-rabbit Peroxidase-labeled antibody (SeraCare, 5220-0336)

Full Protocol:

• Grow 7.5x10⁵ HEK293 cells in DMEM-medium (Sigma-Aldrich, D6429)) supplemented with 10% Fetal Bovine serum (Sigma-Aldrich, F7524) and 1% penicillin-streptomycin (Sigma-Aldrich, P0781), at 37°C and 5% CO₂ in 2ml on a 6-well-dish (Greiner Bio-One) ON.
• Transfect cells with 2μg of respective pcDNA3 construct using self-prepared PEI transfection reagent.
• After 24h lyse the cells in 250μl per well cold RIPA lysis buffer (1 % NP-40, 1mM EDTA, 50mM Tris-HCl pH7.4, 150mM NaCl, 0.25 % Sodium-deoxycholate, PhosSTOP tablet (Roche, 04906845001) and cOmplete tablet (Roche, 04693132001) diluted in RIPA following the manufacturer´s instructions.
• Denature the cleared lysate of total protein for 5min at 95°C in 50μl of 6x SDS sample buffer and subsequently separate 4μl of each sample on a 10% polyacrylamide gel.
• Transfer proteins onto nitrocellulose membrane (Biostep 01-14-101) with a Tank Blotting System at 10V ON.
• Block the membrane with 1x Casein Blocking Buffer (Sigma-Aldrich, B6429) for 1h at RT with gentle shaking.
• Wash membrane 3x for 10min with TBST.
• Cut the membrane and incubate fragments separately with primary
• • rabbit anti-GRK6 antibody (ABIN3042435, antibodies-online, lot 0971512Da9609109) diluted 1:1000 in 5% BSA-TBST at 4°C ON.
  • rabbit anti-GRK6 antibody (Cell Signaling Technology, 5878, lot 1) diluted 1:1000 in 5% BSA-TBST at 4°C ON.
  • rabbit anti-Vinculin antibody (Biozol, BZL03106, lot 0401) diluted 1:1000 in 5% BSA-TBST at 4°C ON.
• Wash membrane 3x for 10min with TBST.
• Incubate membrane with secondary goat-anti-rabbit Peroxidase-labeled antibody (SeraCare, 5220-0336) diluted 1:10000 in 1x Casein Blocking Buffer) for 1h at RT with gentle shaking.
• Wash membrane 3x for 10min with TBST.
• Reveal protein bands using Western Lightning Plus ECL reagent (Perkin Elmer, NEL103001EA, Luminol reagent Lot 275-17431, Oxidizing reagent, lot 265-17431) on a LAS-4000 Luminescence Imager (Fujifilm), exposure for 15 and 90 seconds.

Experimental Notes:

If the detection of the smallest isoform (GRK6-4, transcript variant 4, NM_001364164.1) is required, ABIN3042435 is a good choice, as this isoform is not detected by the alternative GRK6 antibody tested in parallel. Nevertheless, the strong background signal around 55kDa produced by ABIN3042435 diminishes its usefulness.

Handling

Format: Lyophilized

Reconstitution: Add 0.2 mL of distilled water will yield a concentration of 500 μg/mL.

Concentration: 500 μg/mL

Buffer: Each vial contains 4 mg Trehalose, 0.9 mg NaCl and 0.2 mg Na2HPO4.

Handling Advice: Avoid repeated freezing and thawing.

Storage: 4 °C,-20 °C

Storage Comment: Store at -20°C for one year from date of receipt. After reconstitution, at 4°C for one month.
It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freeze-thaw cycles.

Expiry Date: 12 months

References for anti-GRK6 antibody (ABIN3042435)

Reichel, Weitzel, Klement, Hoffmann, Drube: "Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots." in: International journal of molecular sciences, Vol. 23, Issue 3, (2022) (PubMed).

Target Details for GRK6

Target: GRK6 (G Protein-Coupled Receptor Kinase 6 (GRK6))

Alternative Name: GRK6

Background:

Background: G protein-coupled receptor kinase 6?is an?enzyme?that in humans is encoded by the?GRK6?gene. It is mapped to 5q35. This gene encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase subfamily of the Ser/Thr protein kinase family. The protein phosphorylates the activated forms of G protein-coupled receptors thus initiating their deactivation. Several transcript variants encoding different isoforms have been described for this gene. Also, GRK6 appears to be involved in responses to?morphine.

Gene Full Name: G protein-coupled receptor kinase 6

Molecular Weight: 66 kDa

Gene ID: 2870

UniProt: P43250

Pathways: Myometrial Relaxation and Contraction, Regulation of G-Protein Coupled Receptor Protein Signaling, CXCR4-mediated Signaling Events, Negative Regulation of Transporter Activity