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).

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: 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

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).

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