Anti-RbcS | Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Algal)

5 µg of total protein from C.reinhardtii was denatured with Invitrogen LDS sample buffer (4X) at 70°C/5 min.  Samples were separated on Invitrogen NuPage Bis-Tris 4-12% SDS-PAGE 60 min and blotted for 1 h to Invitrogen PVDF (pore size of  0.45  µm), using wet transfer. Blot was blocked with 5% milk in TBS-T for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1h/RT with TBS-T Blocking. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 10 min and 2 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG ALP conjugated, AS09 607 lot 2302) diluted to 1: 5 000 in TBS-T Blocking for 0.5h/RT with agitation. The blot was washed as above and developed with AS19 BCIP-NBT for 30 seconds. Image was captured after 1h.

Samples: 
1-4 Arabidopsis thaliana leaf tissue
5-8  Arabidopsis thaliana soluble extract from leaf tissue

40 mg of fresh Arabidopsis thaliana leaf tissue was used for extraction of the protein. To 40 mg of the tissue was resuspended in 400 µl of extraction buffer. Extraction buffer consists of following components: 100mM EDTA pH8.0, 120mM Tris-HCl pH 6.8, 4%w/v SDS, 10% v/v ß-Mercaptoethanol, 5% v/v glycerol, 0,005% w/v Bromphenol blue. Leaf tissue was ground in mortar and pestle in the Protein extraction Buffer. Boiled for 5 minutes at 95°C, span 5 min at top spin of table-top centrifuge (21.000g) supernatant used for PAGE. From the extract prepared from wt Col-0 a dilution series was prepared and loaded onto the gel. 5 µl, 2,5µl, 1,25µl and 0,62µl were used for the Antibody test. Next to the total protein extract soluble fraction of the cells extracted under native conditions in following buffer were used: 450 mM solbitol, 20mM Tricine-KOH pH8.4, 10mMEDTA pH8.0, and 0,1%BSA. From that extract concentration 3µg/µl a dilution series was loaded as following: 5µl, 2,5µl, 1,25µl, and 0,62µl. The lowest concentration used for detection range is 1,86µg of soluble protein. Samples were separated at RT PAGE on 12% SDS-PAGE and blotted with semidry blotting machine (BioRad) for 30 min to nitrocellulose membrane (Merck). Blot was blocked in 4%Milk for 1h at RT with agitation. Blot was incubated ON in cold in the primary antibody diluted 1:2000 with agitation in 2%Milk TBS-T solution. The antibody solution was decanted, and the blot was rinsed 4 times a 5 to 10 minutes in 1xTBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated antibody) which was diluted 1:10.000. The incubation was done for 1h at RT with agitation. The blot was washed as above and developed with self-made developing solution. Exposure time was 3 minutes.