CLNS1A antibody

Catalog No. ABIN933127

Product Details anti-CLNS1A Antibody

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

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This CLNS1A antibody is un-conjugated

Application: Western Blotting (WB), Immunocytochemistry (ICC), Dot Blot (DB)

Cross-Reactivity: Human, Mouse (Murine), Rat (Rattus)

Cross-Reactivity (Details): Other species not studied.

Purification: Protein G affinity chromatography

Immunogen: CLNS1 A antibody was raised in mouse using recombinant Human Chloride Channel, Nucleotide-Sensitive, 1 (Clns1 )

Clone: 2187C2a

Isotype: IgG1

Application Details

Application Notes: WB: 0.2-2 µg/mL, ICC: 2-100 µg/mL
Optimal conditions should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #102907 (Western Blotting)

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

No.: #102907

Date: 09/20/2018

Antigen: CLNS1A

Lot Number: 9832

Method validated: Western Blotting

Positive Control:

HeLa cells

Independent antibody

Negative Control:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Notes:

Passed. ABIN933127 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 ABIN933127 (A) and an independent antibody (B). Tubulin serves as loading control (C).

Full Methods

Primary Antibody: ABIN933127

Secondary Antibody: goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, 115-005-003, lot 128525)

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 2 ml media.
• Grow for 24h cells 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
• • mouse anti-CLNS1A antibody (antibodies-online, ABIN933127, lot 9832) 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
• • goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, 115-005-003, lot 128525) diluted 1:10000 in PBS for 45min at RT or
  • goat anti-rat IgG (H+L) (Jackson ImmunoResearch, 112-035-003) diluted 1:10000 in PBS for 45min at RT.
• Wash membrane 5x for 5min with 1x 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.

Independent Validation (IF)

Validation #103452 (Immunofluorescence)

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

No.: #103452

Date: 09/20/2018

Antigen: CLNS1A

Lot Number: 9832

Method validated: Immunofluorescence

Positive Control:

HeLa cells

Independent antibody

Negative Control:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Notes:

Passed. ABIN933127 specifically reveals endogenous CLNS1A in HeLa cells while no signal is detected subsequently to knock-down of the antigen.

Validation Images

Immunofluorescence labeling of CLNS1A in HeLa cells (HeLa) using ABIN933127 (A) or an independent antibody (B). The signal disappears after CLNS1A knock-down (HeLa/kd).

Full Methods

Primary Antibody: ABIN933127

Secondary Antibody: AF488 conjugated secondary goat anti-mouse IgG (H+L) (ThermoFisher Scientific, A32723)

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.
• Transfer cells from each well on five coverslips.
• Grow cells for 96h at 37°C and 5% CO₂.
• Wash cells with 1x PBS (PAN Biotech).
• Add 4% PFA for 10min at 37°C.
• Wash cells 2x with 1x PBS 1x.
• Wash cells 1x 5min with 0.5% Triton X-100.
• Wash cells 2x with 1x PBS containing 1x Roti-Immunoblock (Roth, T144.1, lot 248272501).
• Store cells ON at 4°C.
• Incubation with primary
• • mouse anti-CLNS1A antibody (antibodies-online, ABIN933127, lot 9832) diluted 1:500 in Roti-Immunoblock for 1h at RT or
  • mouse anti-CLNS1A antibody (Santa Cruz, sc-271454, G3010) diluted 1:500 in Roti-Immunoblock for 1h at RT.
• Wash coverslips with PBS.
• Incubation with AF488 conjugated secondary goat anti-mouse IgG (H+L) (ThermoFisher Scientific, A32723) diluted 1:1000 for 45min at RT.
• Wash coverslips with PBS.
• Mount coverslips on glass slides in Fluoromount-G mounting medium (Southern Biotech, 0100-01).
• Image acquisition with an exposure time of 600ms (ABIN933127) or 400ms (sc-271454) on (Zeiss Axiophot, 63x magnification, resolution 1388x1040 pixels).

Experimental Notes:

IF staining of HeLA cells using ABIN933127 reveals the expected staining pattern. Cellular staining was not observed after CLNS1A knock-down.

Handling

Concentration: Lot specific

Buffer: CLNS1 A antibody in PBS (3.0 mM KCl, 1.5 mM KH2 PO4 , 140 mM NaCl, 8.0 mM Na2 HPO4 (pH 7.4)) containing 1 % bovine serum albumin (BSA) and 0.05 % sodium azide (NaN3 ).

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.
Dilute only prior to immediate use.

Storage: 4 °C/-20 °C

Storage Comment: Store at 2-8 °C for up to one year. We recommend long term storage at -20 °C.

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: The interaction with Sm proteins inhibits their assembly on U RNA and interferes with snRNP biogenesis. Inhibits the binding of survival motor neuron protein (SMN) to Sm proteins. May participate in cellular volume control by activation of a swelling-induced chloride conductance pathway. Synonyms: Monoclonal CLNS1A antibody, Anti-CLNS1A antibody, Chloride channel nucleotide sensitive 1A antibody, CLCI antibody, ICln antibody, CLNS1B antibody.

Pathways: Ribonucleoprotein Complex Subunit Organization