Anti gfp antibody

To look at the protein level of wild type and mutants of Sbp1 in BG1805 construct, cells were first grown in SD ura-minimal media with glucose till 0.45–0.5 OD600 this was followed by pelleting and growing in SD ura- 2% galactose overnight. Cells were broken open using acid wash glass beads and 20microgram of total protein was loaded in 8% SDS polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane using Bio-Rad wet transfer apparatus. Post transfer, the membrane was stained with Ponceau S to know the total protein present in each lane. The blot was washed and blocked using skimmed milk. PAP (1:5000, Sigma Aldrich cat# P1291) was used to detect over expressed Sbp1 and mutant proteins. Sbp1-GFP and its mutants protein level was looked at the same way as gal inducible Sbp1 except the use of anti-GFP anti body (1: 1000, BioLegend cat# 902602). For loading control, blot was stripped and put in anti - PGK1 antibody (1:1000, Abcam cat# AB113687).

Proteins were extracted from frozen and homogenized seeds. For 4 mg seed material, 100 µL freshly prepared extraction buffer (4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 2 mM phenylmethane sulfonyl fluoride, 0.1 M Tris pH 8.5) was added. Samples were immediately, vigorously vortexed for at least 2 min. Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20,810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4 × Läemmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4 × Läemmli buffer diluted protein extract was loaded on an SDS gel. For western blot analysis, proteins were detected using an anti-GFP antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-alkaline phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.

Seedlings were grown for 10 days in a vertical orientation on half-strength MS plates, and 100 seedlings were collected and grinded in liquid nitrogen. Proteins were extracted with 200 μl 1% SDS, boiled for 5 minutes, cooled on ice, centrifuged, and 20 μl were mixed with 5 μl 5x Lämmli buffer for loading on a 10% SDS-PAGE gel (BioRad). After blotting was done on nitrocellulose using the semi-dry system (BioRad), the membrane was blocked overnight with 1xTBST, 5% non-fat dry milk powder. Immunolabeling was done in 1xTBST, 1% non-fat dry milk powder with a 1:3’000 dilution of an anti-GFP antibody (Biolegend, 902601), followed by a 1:5’000 dilution of an anti-mouse-HRP antibody (Sigma Aldrich, A 4416).