CLNS1A anticorps

N° du produit ABIN4903315

Cet validé indépendamment anticorps Lapin Polyclonal détecte spécifiquement CLNS1A dans WB, FACS et IHC. Il présente une réactivité envers Humain.

Aperçu rapide pour CLNS1A anticorps (ABIN4903315)

Antigène: CLNS1A (Chloride Channel, Nucleotide-Sensitive, 1A (CLNS1A))

Reactivité: Humain

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp CLNS1A est non-conjugé

Application: Western Blotting (WB), Flow Cytometry (FACS), Immunohistochemistry (IHC)

Détail du produit anti-CLNS1A anticorps

Purification: Affinity purification

Immunogène: A synthetic Peptide of human CLNS1A

Isotype: IgG

Information d'application

Indications d'application: WB 1:500 - 1:1000
IHC 1:20 - 1:50
FC 1:20 - 1:50

Restrictions: For Research Use only

Independent Validation (WB)

Validation #102909 (Western Blotting)

by: AG Gruss, Institut für Genetik, Rheinische Friedrich-Wilhelms-Universität Bonn

No.: #102909

Date: 20.09.2018

Antigène: CLNS1A

Numéro du lot: 03R181011

Application validée: Western Blotting

Contrôle positif:

HeLa cells

Independent antibody

Contrôle négative:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Conclusion:

Passed. ABIN4903315 specifically reveals endogenous CLNS1A in HeLa cell lysates while no signal is detected subsequently to knock-down of the antigen.

Validation Images

Western blot analysis of HeLa cell lysates without (HeLa) or with siRNA knock-down (HeLa/kd). CLNS1A was specifically detected using ABIN4903315 (A) and an independent antibody (B). Tubulin serves as loading control (C).

Protocole

Anticorps primaire: ABIN4903315

Anticorps secondaire: HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, 111-035-003)

Full Protocol:

• Seed 3x10⁴ HeLa cells in DMEM (PAN Biotech, P04-41450, 2730618) supplemented with 10% FBS (PAN Biotech, P03-3302), without antibiotics, in 6 well plates in 2ml media.
• Grow cells for 24h at 37°C and 5% CO₂ to 70% confluency.
• Change medium.
• Transfect cells with 60pmol ON-TARGETplus SmartPool siRNA human (Dharmacon, L-012571-00-0005, lot 170831) in 125µl 150mM NaCl added to 7.5µl Lipofectamine RNAiMax (13778030, lot 1911816) in 125µl 150mM NaCl.
• Grow cells for 24h at 37°C and 5% CO₂.
• Change medium.
• Grow cells for 72h at 37°C and 5% CO₂.
• Wash cells once with PBS (PAN Biotech, P04-36500, lot 3620618).
• Detach cells with 100µl trypsin per well.
• For WB the cells were once washed with PBS (PAN Biotech) before treated with 100µl of Trypsin (PAN Biotech, P10-023100, lot 6860417) per well.
• Once cells are detached add 900µl medium.
• Count cells.
• Add 40µl 1x Laemmli buffer (Roth, K929.1, lot 227259243) to 2x10⁵ cells. Heat up to 99°C for 10min.
• Separate 10µl of the lysates on a freshly cast denaturing 12% SDS-PAGE gel in an electrophoresis chamber for 1h at 120V.
• Transfer proteins onto PVDF membrane (Merck, IPVH0010, lot K5KN7128I) with a wet blotting system for 1h at 80V.
• Block the membrane with 1x Roti Block (Roth, A151.2, lot 148270222) for 30min at RT.
• Incubation with primary
• • rabbit anti-CLNS1A antibody (antibodies-online, ABIN4903315, lot 03R181011) diluted 1:500 in 1x Roti Block for 1h at RT,
  • mouse anti-CLNS1A antibody (Santa Cruz, sc-271454, G3010) diluted 1:3000 in 1x Roti Block for 1h at RT, or
  • rat anti-tubulin antibody (YOL 1/34 gifted) diluted 1:1000 in 1x Roti Block for 1h at RT.
• Wash membrane 2x for 10min with 1x TBST.
• Incubation with secondary
• • HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, 111-035-003) diluted 1:1000 in 1x Roti Block for 1h at RT,
  • goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, 115-005-003, lot 128525) diluted 1:10000 in PBS for 45min at RT, or
  • HRP-conjugated goat anti-rat IgG (H+L) (Jackson ImmunoResearch, 112-035-003) diluted 1:10000 in PBS for 45min at RT.
• Wash membrane 5x for 5 min with TBS-T.
• Reveal protein bands using ECL (GE Healthcare, RPN3244V2, 9633722) on an LAS mini system (GE healthcare).

Notes:

Western blot using ABIN933127 on HeLa cell extracts reveals a band of the expected molecular weight. The same band is also detected by an independent CLNS1A antibody and disappears upon siRNA knock-down.

Stockage

Format: Liquid

Buffer: PBS with 0.02 % sodium azide, 50 % glycerol,  pH 7.3.

Agent conservateur: Sodium azide

Précaution d'utilisation: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Conseil sur la manipulation: Avoid repeated freeze/thaw cycles.

Stock: -20 °C

Stockage commentaire: Store at -20C. Avoid freeze / thaw cycles.

Détails sur CLNS1A

Antigène: CLNS1A (Chloride Channel, Nucleotide-Sensitive, 1A (CLNS1A))

Autre désignation: CLNS1A

Sujet: This gene encodes a protein that functions in multiple regulatory pathways. The encoded protein complexes with numerous cytosolic proteins and performs diverse functions including regulation of small nuclear ribonucleoprotein biosynthesis, platelet activation and cytoskeletal organization. The protein is also found associated with the plasma membrane where it functions as a chloride current regulator. Pseudogenes of this gene are found on chromosomes 1, 4 and 6. Several transcript variants encoding different isoforms have been found for this gene.

Poids moléculaire: 26.215 kDa

ID gène: 1207

UniProt: P54105

Pathways: Ribonucleoprotein Complex Subunit Organization