AS07 225 Anti-A12,2 | RNA polymerase I subunit (homolog of Pol II Rpb9)

Confirmed reactivity:	Arabidopsis thaliana
Predicted reactivity:	Arabidopsis thaliana
Not reactive in:	No confirmed exceptions from predicted reactivity are currently known

10 µg of total protein from (1) Arabidopsis thaliana and (4) Oryza sativa leafs extracted with PEB (AS08 300), as well as (2) cytosolic and (3) nuclear fractions of Arbaidopsis thaliana leafs were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to nitrocellulose. Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-A12.2 (AS07 255, 1:1000, 1h) and secondary anti-rabbit (1:20 000, 1 h) antibody (HRP conjugated) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T (15, +5, +5, +5 min). All steps were performed at RT with agitation. Signal was detected with chemiluminescent reagent using a Fuji LAS-3000 CCD (300s, standard sensitivity). The target A12.2 is specifically detected only in the nuclear extract of Arabidopsis thaliana (3).

Additional information:	This product has previously been labelled as anti-At3g25940 transcription factor S-II (TFIIS) domain-containing protein. Protocol for isolation of cytosolic and nuclear fractions can be found here.
Additional information (application):	This antibody is specific for A12,2 subunit of RNA polymerase I but NOT RNA polymerase II or IV from Arabidopsis thaliana

RNA polymerase I is a nuclear located DNA-dependent enzyme involved in RNA elongation and regulation of transcription. In yeast subunit A12.2 has been described as homologous to the Rpb9 subunit from polymerase II.

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

 

Preparation of cytosolic and nuclear protein fractions

1. Prepare protoplasts from 50 ml Arabidopsis thaliana cell culture according to the protocol of PEG transfection.
2. Resuspend protoplasts in 10 ml GH buffer and keep the solution on ice for 10 min.
GH buffer: 100mM glycine
0.1% Hexylene glycol
0.37M (4.7% w/v) saccharose
0.3mM Spermine
1.0mM Spermidine
pH 8.3 with Ca(OH)2
3. To release nuclei add Triton X100 to a final concentration of 0.1%. Pipetting gently up and down several
times with a plastic pipette might be necessary to lyse cells.
4. After 5min sediment nuclei by centrifugation at 1000 xg for 15 min at 4°C. Save supernatant as the
cytoplasmic fraction. Wash the pelleted nuclei two times with GHT (GH+0.1% TX100) then finally
resuspended in a suitable volume of extraction buffer + protease inhibitors.

Courtesy Dr. Laszlo Bako, Umeå Plant Science Centre