Application
Western blot
Sample
Rat Tissue lysate - whole (P90 Cerebellum)
Gel Running Conditions
Reduced Denaturing (8% gel)
Loading amount
100 µg
Specification
P90 Cerebellum
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 0.1% Tween 20 -TBS (T-TBS)
Dilution
1/25000
Secondary antibody
Dilution
1/50000
Name
Non-Abcam antibody was used: Anti-mouse antibody (Jackson Immuno Research Inc.)
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL
Negative control
Omission of primary or secondary antibodies.
Exposure
4 minute(s) and 0 second(s)
Bands
Specific: 95-108-110 kDa
Positive control
GABA B2: 106 kDa
Additional data
Additional Notes
Protein extracts from whole rat cerebellum were generated in a three-step procedure using Triton Lysis Buffer [LB: 0.5% (v/v) Triton X-100, 150mM NaCl, 5mM EDTA, 1M Tris-HCl, pH 7.5), supplemented with phosphatase and protease inhibitors [0.01% NaF and 0.001% Protease Inhibitor Cocktail (P8340 Sigma-Aldrich), respectively]. Samples were heated at 50¯C for 5 minutes and then cooled down gradually at room temperature (RT) for 45 minutes. This step ensures protein denaturalization while preventing the oligomerization of highly hydrophobic residues present in the typical seven transmembrane spanning regions of G protein–coupled receptors (GPCRs). Proteins were next separated by 8% SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane by semi-dry electroblotting using Towbin Buffer with 0.1% SDS and 10% methanol, and incubated, after methanol activation, with blocking solution [5% (w/v) of skim milk in TBS with 0.1% Tween-20 (T-TBS) for GABA B1 5% (w/v) of skim milk in PBS with 0.1% Tween-20 (T-PBS) for GABA B2 and actin] for 1 hour at RT. Figure: A) Increasing dilutions for GABA B1 antibody at different protein concentrations of whole cerebellar extract. The three bands of 95, 108 and 110 KDa correspond to the GABA B1 isoforms 1b, 1a and 1f, respectively. B) Lanes 1 to 7: Ponceau staining of the PVDF membrane after semi-dry transference. C) Representative blots of GABA B receptor 1, GABA B receptor 2 and actin in an enriched protein extract from whole rat cerebellum. Lane 1: protein ladder (PageRuler Plus Prestained, Thermo Scientific). Lane 2: 50 ´g of whole protein extract: GABA B receptor 2 (106 KDa) was used as positive control (for more information regarding the antibody ab75838 see abreview 62172: https://www.abcam.com/GABA-B-Receptor-2-antibody-EP2411Y-ab75838/reviews/62172). Lanes 3 to 5: 10, 50, and 100 ´g of whole protein extract: two isoforms of the subunit B1: 1a (108 KDa) and 1b (95 KDa), are observed. Lanes 6 and 7: 50 ´g negative controls by omission of the anti-GABA B receptor 1 antiserum and the secondary antibody, respectively. In lanes 2 to 7, actin (42 KDa) is also shown as loading control. MW: molecular weight. Figure generated by Elena Vàsquez and Estela Muðoz, IHEM, UNCuyo, CONICET, Mendoza, Argentina.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
DR. Estela Munoz
Verified customer
提交于 Dec 29 2017