ACT | Actin (monoclonal, clone 14H4G8), 10 µg

10 µg/well of total protein extracted freshly from Arabidopsis thaliana leaf tissue. All lanes shown are from different Arabidopsis thaliana leaf samples extracted simultaneously. Fresh leaf tissue was ground up directly in 1x Bolt LDS loading buffer (Thermo) and 200mM DTT and denatured at 80°C for 10 min. Samples were separated on 12% SDS-PAGE gel and transferred to nitrocellulose by wet transfer for 1hr at 100V. Blot was blocked with 5 % milk in TBST for 1h/RT with agitation. Blot was incubated with Agrisera Mouse anti-actin monoclonal (AS21 4615) at a dilution of 1:1000, in 5% milk with agitation at 1h RT. The blot was rinsed 3 times for 5 minutes in TBS-T with agitation. Then the membrane was incubated with secondary anti-mouse HRP (Agrisera AS09 627) at 1:10 000 dilution in milk for 1hr at room temp. The blots were washed as above and reaction was visualized using ECL reagent and following manufacture's recommendations. The actin band was visualized after 15 seconds of film exposure.



Immunofluorescent localization of actin on suspension culture of Oryza sativa ssp. japonica cv. 'Unggi 9', using anti actin (AS21 4615) and anti-mouse IgG DyLight® conjugated secondary antibodies (AS10 1261). Few representative actin filaments are highlighted by yellow arrowheads. DAPI staining of nuclei is pseudocolored red. 

Material: Suspension cultures of Oryza sativa ssp. japonica cv. 'Unggi 9'
Fixation: Packed cell volume to fixer ratio: 250 µl : 5ml
Fixer composition and buffer: 4% (w/v) paraformaldehyde (freshly prepared as 8% stock and 0.2 µm filtered) in Phosphate Buffered Saline (PBS), pH 7.4 (2x stock, 0.2 µm filtered)
Container and method: in 6 cm Petri dish, gentle shaking at room temperature (RT)
Duration: 40 minutes. Triton X100 is not used in fixer. Cells were not shaken during the first 5 mins of fixation to allowed to partially recover from osmotic shock induced by formaldehyde.
Hydrophilization: no
Cell wall digestion: Yes Packed cell volume to enzyme ratio: 100ul : 2ml Enzyme composition: 1.2% Cellulase (chromatically purified, powder, Worthington), 1.2% (R) Pectinase (protease free, liquid, Sigma) Buffer: 0.5% (w/v) MES buffer, pH 5.6
Container and method: in 2 ml microfuge tube by rolling at room temperature (RT)
Duration:  60 minutes
Membrane permeabilization: Triton-X100 (0.35%), 7 min/RT
Antigen retrieval: no
Blocking buffer: Fish gelatin (5% v/v)
Washing buffer: PBS
Primary antibody dilution and incubation time: 1:500, 1hr/RT
Secondary antibody dilution and incubation time and supplier: DyLight® 488 (AS10 1261) 1:600, 45 min/RT
Co-staining of the nucleus (DAPI): Yes
Nucleus staining: 100 ng/ml DAPI

Courtesy of Dr. Ferhan Ayaydin, Hungarian Centre of Excellence for Molecular Medicine (HCEMM), Szeged, Hungary.