GST-Tag Antikörper

Produktnummer ABIN3045984

Produktdetails anti-GST-Tag Antikörper

Target: GST-Tag

Reaktivität: Schistosoma japonicum

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser GST-Tag Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Chromatin Immunoprecipitation (ChIP)

Produktmerkmale: The GST tag issued in many protocols

Aufreinigung: purified form hybridoma supernatant

Immunogen: GST

Klon: 5C12

Isotyp: IgG1

Anwendungsinformationen

Applikationshinweise: WB 0.1-1.0ug/ml

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #029860 (Western Blotting)

by: Celplor LLC

No.: #029860

Datum: 19.05.2016

Antigen: Mouse anti-GST monoclonal antibody

Chargennummer: 1214

Validierte Anwendung: Western Blotting

Positivkontrolle: BL21 bacteria cells were transformed with an in-house GST expression vector pGST

Negativkontrolle: Empty vector cell lysate

Bewertung: Based on the 12% GST content in total cell lysate of BL21-pGST (+IPTG), there is an estimated 1 ng of GST protein in 0.01 ug of total protein with IPTG induction. Therefore, the detection limit of mouse anti-GST monoclonal antibody is lower than 1 ng of GST protein.

Validierungsbilder

Figure 1: GST content of BL21-pGST(+IPTG) is 12% of total protein. GST content in IPTG induced samples was analyzed by Quantity One software (Bio-Rad).

Figure 2: Western blot for mouse anti-GST monoclonal antibody. The first lane shows the empty vector cell lysate (negative control). This is followed by a 0.01, 0.1, and 1 ug of total protein from BL21-pGST, with (Lanes 2, 3, and 4) or without (Lanes 5, 6, and 7) IPTG induction, respectively. Based on the 12% GST content in total cell lysate of BL21-pGST (+IPTG), there is an estimated 1 ng of GST protein in 0.01 ug of total protein with IPTG induction. Therefore, the detection limit of mouse anti-GST monoclonal antibody is lower than 1 ng of GST protein. The higher than expected molecular weight may be the result GST dimers or aggregates (Riley et al. Protein Engineering vol.9 no.2 pp.223-23O, 1996).

Protokoll

Primärantikörper:

• Antibody: Mouse anti-GST monoclonal antibody
• Lot number: 1214
• Dilution: 1:1000

Sekundärantikörper:

• Antibody: HRP labeled secondary anti-mouse antibody
• Lot number: 390630
• Dilution: 1:2000

Full Protocol:

• **I. Reagent preparation**
• 1. BL21 bacteria cells were transformed with an in-house GST expression vector pGST.
• 2. Individual colony was inoculated in 2xYT medium and cultured at 37°C with shaking for 5 hrs followed by IPTG (1mM) induction for 2 hrs. Bacteria was grown to OD600=0.6-0.8 (5 hr) when IPTG was added.
• 3. Cells were centrifuged at top speed and pellets were collected.
• 4. Cell pellets were lysed in SDS-PAGE sample buffer.
• 5. Protein concentration was assayed using DC protein assay kit (Bio-Rad).
• 6. 50 ug of cell lysate was loaded and protein was separated by SDS-PAGE.
• 7. Gel was stained with SimplyBlue Safe Stain (Invitrogen) at room temperature.
• 8. GST content in IPTG induced samples was analyzed by Quantity One software (Bio-Rad).
• **II. Western blot validation**
• 1. Cell lysates from BL21 host cells, BL21-pGST transformed cells with or without IPTG induction were loaded and proteins were separated by SDS-PAGE followed by Western transfer to a nitrocellulose membrane.
• 2. Membrane was blocked with 2% powder milk (Bio-Rad) for 1 hr at room temperature.
• 3. Membrane was incubated with mouse anti-GST monoclonal antibody (1:1000 dilution) at 4°C overnight with shaking.
• 4. Membrane was washed with TBST three times for 10 min.
• 5. After washing, membrane was incubated with HRP labeled secondary anti-mouse antibody (1:2000 dilution, GE Healthcare) with shaking at room temperature for 1 hr.
• 6. Membrane was washed with TBST three times for 10 min.
• 7. After washing, membrane was incubated with SuperSignal West Pico substrate (Thermo Scientific) for 5 min at room temperature.
• 8. Membrane was analyzed by ChemiDoc XRS gel documentation system (Bio-Rad).

Handhabung

Format: Liquid

Konzentration: 1 mg/mL

Buffer: PBS pH7.2

Lagerung: -20 °C

Details zu GST-Tag

Target: GST-Tag

Andere Bezeichnung: GST Tag (GST-Tag Produkte)

Substanzklasse: Tag

Molekulargewicht: 26 kDa

UniProt: P08515