CLNS1A anticorps

N° du produit ABIN933127

Détail du produit anti-CLNS1A anticorps

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

Reactivité: Humain

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp CLNS1A est non-conjugé

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

Réactivité croisée: Humain, Souris, Rat (Rattus)

Réactivité croisée (Details): Other species not studied.

Purification: Protein G affinity chromatography

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

Clone: 2187C2a

Isotype: IgG1

Information d'application

Indications d'application: 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: 20.09.2018

Antigène: CLNS1A

Numéro du lot: 9832

Application validée: Western Blotting

Contrôle positif:

HeLa cells

Independent antibody

Contrôle négative:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Conclusion:

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).

Protocole

Anticorps primaire: ABIN933127

Anticorps secondaire: 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).

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: 20.09.2018

Antigène: CLNS1A

Numéro du lot: 9832

Application validée: Immunofluorescence

Contrôle positif:

HeLa cells

Independent antibody

Contrôle négative:

HeLa cells, CLNS1A knockdown using Dharmacon smartpool siRNA

Conclusion:

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).

Protocole

Anticorps primaire: ABIN933127

Anticorps secondaire: 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).

Notes:

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

Stockage

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 ).

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.
Dilute only prior to immediate use.

Stock: 4 °C/-20 °C

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

Détails sur CLNS1A

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

Autre désignation: CLNS1A (CLNS1A Produits)

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

Sujet: 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