CLNS1A antibody

Catalog No. ABIN4903315

Product Details anti-CLNS1A Antibody

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

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This CLNS1A antibody is un-conjugated

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

Purification: Affinity purification

Immunogen: A synthetic Peptide of human CLNS1A

Isotype: IgG

Application Details

Application Notes: 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: 09/20/2018

Antigen: CLNS1A

Lot Number: 03R181011

Method validated: Western Blotting

Positive Control:

HeLa cells

Independent antibody

Negative Control:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Notes:

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).

Full Methods

Primary Antibody: ABIN4903315

Secondary Antibody: 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).

Experimental 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.

Handling

Format: Liquid

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

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 -20C. Avoid freeze / thaw cycles.

Target Details for CLNS1A

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

Alternative Name: CLNS1A (CLNS1A Products)

Synonyms: CLCI antibody, CLNS1B antibody, ICln antibody, 2610036D06Rik antibody, 2610100O04Rik antibody, Clci antibody, Clcni antibody, ICLN antibody, Icln antibody, pICln antibody, clci antibody, clns1b antibody, icln antibody, pIcIn antibody, chloride nucleotide-sensitive channel 1A antibody, chloride channel, nucleotide-sensitive, 1A antibody, chloride channel, nucleotide-sensitive, 1A L homeolog antibody, CLNS1A antibody, Clns1a antibody, clns1a.L antibody, clns1a antibody

Background: 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.

Molecular Weight: 26.215 kDa

Gene ID: 1207

UniProt: P54105

Pathways: Ribonucleoprotein Complex Subunit Organization