Reactivité: Tag
WB, ELISA
Hôte: Lapin
Polyclonal
unconjugated
Indications d'application
WB 0.1-1.0ug/ml
Restrictions
For Research Use only
Validation #029860
(Western Blotting)
by
Celplor LLC
No.
#029860
Date
19.05.2016
Antigène
Mouse anti-GST monoclonal antibody
Numéro du lot
1214
Application validée
Western Blotting
Contrôle positif
BL21 bacteria cells were transformed with an in-house GST expression vector pGST
Contrôle négative
Empty vector cell lysate
Conclusion
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.
Validation Images
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).
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).