SLC1A3 antibody (AA 522-541)

Catalog No. ABIN2690503

This independently validated Rabbit Polyclonal antibody specifically detects SLC1A3 in WB, IHC, IP and ICC. It exhibits reactivity toward Human, Rat and Mouse.

Quick Overview for SLC1A3 antibody (AA 522-541) (ABIN2690503)

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

Reactivity: Human, Rat, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This SLC1A3 antibody is un-conjugated

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

Grade: KO Validated

Product Details anti-SLC1A3 Antibody

Binding Specificity: AA 522-541

Purpose: anti-EAAT1

Cross-Reactivity (Details): Reacts with: human (P43003), rat (P24942), mouse (P56564). Other species not tested yet.

Purification: affinity purified

Immunogen: Synthetic peptide corresponding to AA 522 to 541 from rat EAAT1 (UniProt Id: P24942)

Application Details

Application Notes: WB:1 : 1000 up to 1 : 10000 , IP:yes ICC:1 : 1000 up to 1 : 5000, IHC:1 : 1000 up to 1 : 5000, IHC-P:not tested 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 50 μLH2O to get a 1 mg/mL solution in PBS.

Buffer: PBS. Albumin was added for stabilization.

Handling Advice: Do not freeze! Antibodies should be stored at +4°C when still lyophilized.

Storage: -20 °C,-80 °C,4 °C

Storage Comment: Antibodies should be stored at +4°C when still lyophilized. Then aliquot and store at -20°C to -80°C until use. Albumin was added for stabilization. Do not freeze!

Target Details for SLC1A3

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

Alternative Name: EAAT1

Background: GLAST, SLC1A3, Glutamate Transporter, Neuroinflamm-Astrocyt, GLAST-1

UniProt: P24942

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