SLC1A3 antibody (Cytoplasmic Domain)

Catalog No. ABIN2690503

Product Details anti-SLC1A3 Antibody

Target: SLC1A3 (Solute Carrier Family 1 (Glial High Affinity Glutamate Transporter), Member 3 (SLC1A3))

Binding Specificity: Cytoplasmic Domain

Reactivity: Human, Rat, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This SLC1A3 antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC)

Purification: affinity purified

Application Details

Application Notes: Optimal working dilution should be determined by the investigator. This product is not tested in IP yet.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103349 (Western Blotting)

by: Department of Chemical Biology, Rutgers University

No.: #103349

Date: 08/29/2018

Antigen: SLC1A3

Lot Number: 250113/3

Method validated: Western Blotting

Positive Control:

Sut2 (xCT-/-), MASS20 (xCT+/+) and MelanA (xCT+/+) cells are all mouse melanocytes that express SLC1A3 transporter. Sut2 and MelanA were from Dr. D. Bennett in 1998 and have kept in our lab since then, MASS20 was made in our lab.

Notes:

Passed. ABIN2690503 does specifically recognize SLC1A3 in all three tested murine melanocyte extracts.

Validation Images

Western blot analysis with ABIN2690503 of Sut2 (SUT2), MASS20 (Mass20) and MelanA (MelanA) cells extracts and extracts from Sut2 cells transfected with xCT and/or Myc-Grm1. Image exposure 5s.

Full Methods

Primary Antibody: ABIN2690503

Secondary Antibody: HRP conjugated donkey anti rabbit antibody (Merck Millipore, AP182P, lot 3043559)

Full Protocol:

• Grow Sut2 (xCT-/-), MASS20 (xCT+/+) and MelanA (xCT+/+) cells in RPMI medium (Sigma Aldrich, R8758, lot RNBG5596) supplemented with 10% fetal bovine serum (SAFC, 12303C, lot 16C364) and antibiotics (Penicillin-Streptomycin, Sigma Aldrich, 055M4784V, lot 097M4767V) at 37°C and 5% CO₂ in 60mm tissue culture plates to 80% confluency. Supplement Sut2 cell lines 1M 2-Mercaptoethanol (Sigma Aldrich, SHBF4223V) and TPA (Sigma Aldrich, P1585) 0.1mg/ml. Supplement MelanA cells with TPA.
• Using 1x Laemmli buffer (4x Laemmli Sample Buffer (Bio-Rad, 161-0747, lot 64098129) containing 20%/vol 2-Mercaptoethanol (Sigma Aldrich, SHBF4223V), diluted 1:4 in PBS).
• Collect protein lysates using a cell scraper.
• Heat lysates in a glycerol block for 10min at 99°C. Centrifuge lysates at 14000rpm for 10min at 4°C. Collect supernatant in a 1.5ml microcentrifuge tube and store at -80°C.
• Thaw samples on ice and transfer 20µl of each lysate to a 0.5ml microcentrifuge tube.
• Heat lysates in a glycerol block for 5min at 95°C and place them on ice immediately.
• Separate samples on a freshly cast denaturing 10% SDS-PAGE in a Bio-Rad Mini PROTEAN 3 cell (serial number 525BR 047264) for 2h at 105V alongside protein ladder (Bio-Rad, 61-0374).
• Transfer proteins in transfer buffer (made in house) onto nitrocellulose membrane (GVS Life Sciences, 1215471, lot number 7051342) in another Bio-Rad Mini PROTEAM 3 cell (serial number 525BR 047264) for 2h at 4°C at 160mA.
• Confirm successful protein transfer using Ponceau red.
• Block protein using 100ml blocking buffer (transfer buffer containing 0.25% milk) in a Snap i.d. 2.0 (Merck Millipore, C73105).
• Incubate with primary rabbit anti-SLC1A3 antibody (antibodies-online, ABIN2690503, lot 250113/3) diluted 1:10000 and rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, 25778) diluted 1:200 in blocking buffer ON at 4°C.
• Wash membrane 5x in a Snap i.d. 2.0 with 1x TBST.
• Incubation with secondary HRP conjugated donkey anti rabbit antibody (Merck Millipore, AP182P, lot 3043559) diluted 1:5000 in 0.25% milk (powdered goat milk rehydrated in 1X TBST) for 1h at RT.
• Wash membrane 5x in a Snap i.d. 2.0 with 1x TBST.
• Incubate membrane with 2ml Luminata Forte Western HRP Substrate (Merck Millipore, WBLUF0100, 180333) for 3min at RT. Reveal protein bands on a Syngene Gel Documentation system at 5s, 15s, and 30second exposures.

Experimental Notes:

• ABIN2690503 reveals a band of the expected 60kDa MW in each of the three tested melanocyte extracts. Cell lines transfected with both xCT and Myc-Grm1 appear to have a double band at 60kDa.

• SLC1A3 did not appear to be upregulated in the xCT-/- cell line SUT2.

Handling

Format: Lyophilized

Reconstitution: For reconstitution add 200 μL H2O, then aliquot and store at -20 °C until use.

Buffer: PBS

Handling Advice: Affinity purified antibodies are less robust than antisera, since protease inhibitors are also removed during purification. Hence, storage at 4 °C for prolonged periods (i.e. several weeks), is not recommended.

Storage: -20 °C

Storage Comment: Unlabeled lyophilized antibodies are stable in this form without loss of quality at ambient temperatures for several weeks or even months. They can be stored at 4°C for several years. Lyophilized antibodies must not be stored in the freezer, they may be destroyed!

Target Details for SLC1A3

Target: SLC1A3 (Solute Carrier Family 1 (Glial High Affinity Glutamate Transporter), Member 3 (SLC1A3))

Alternative Name: EAAT 1 (SLC1A3 Products)

Synonyms: AF001784 antibody, CG3747 antibody, Dglt-1 antibody, Dglut-1 antibody, Dmel\\CG3747 antibody, EAAT antibody, EAAT1 antibody, dEAAT antibody, dEAAT1 antibody, dEaat1 antibody, slc1a3 antibody, wu:fb98e05 antibody, wu:fd50b05 antibody, zgc:56661 antibody, zgc:77768 antibody, SLC1A3 antibody, GLAST antibody, GluT-1 antibody, EA6 antibody, GLAST1 antibody, AI504299 antibody, B430115D02Rik antibody, Eaat1 antibody, GLAST-1 antibody, GLU-T antibody, Gmt1 antibody, MGluT1 antibody, Excitatory amino acid transporter 1 antibody, solute carrier family 1 member 3 antibody, excitatory amino acid transporter 1 antibody, solute carrier family 1 (glial high affinity glutamate transporter), member 3a antibody, solute carrier family 1 (glial high affinity glutamate transporter), member 3 antibody, Eaat1 antibody, SLC1A3 antibody, PTRG_00379 antibody, Tsp_02392 antibody, slc1a3a antibody, Slc1a3 antibody

Background: Synonyms: GLAST, SLC1A3

Pathways: Sensory Perception of Sound, Synaptic Membrane, Dicarboxylic Acid Transport