Trans blot turbo transfer system

Agrobacterium tumefaciens GV2260 cultures carrying the pEG101-PvH3s-YFP constructs were infiltrated into young but fully expanded tobacco leaves at a concentration of OD_600nm = 0.5. Leaf disks (1.9 cm diameter) were collected 3 days post inoculation using a cork borer, ground in liquid nitrogen and re-suspended in 100 μl 3 × Laemmli buffer containing 16% β-mercaptoethanol. The tissue was then boiled for 10 min and pelleted at a high speed for 10 min. Twenty micro liters of protein extract was applied to and separated on a 10% SDS-PAGE gel. The proteins were blotted to a PVDF membrane using a Bio-Rad Trans-Blot®Turbo^TM Transfer System. The membrane was blocked with 5% nonfat skim milk in 1 × Tris-saline buffer supplemented with 0.5% Tween 20 (1 × TBST). Next, the membrane was probed with anti-HA-HRP (Abcam, 1:2000) and the signal was detected with using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). The chemiluminescent signals were exposed to autoradiography film (Genesee Scientific, San Diego, CA) using a Kodak film processor (Kodak, A Walsh Imaging, Inc, Pompton Lakes, NJ).

Protein extraction was performed using M-PER reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions to lyse the cells, and protein concentrations were measured with DC Protein Assay (Bio-Rad); 15–20 µg of a protein sample was loaded into the precast 10% Mini-PROTEAN® TGX Stain-Free^TM Gels (Bio-Rad). After the electrophoresis run, Trans-Blot® Turbo^TM Pack (Bio-Rad) was used to transfer the proteins from the gel to the nitrocellulose membrane. Transfer was done with the Trans-Blot® Turbo^TM Transfer system according to the manufacturer’s instructions (Bio-Rad). A prestained protein ladder, PageRuler Plus (#26619, Thermo Fischer Scientific), was used as a protein size marker. We utilized antibodies against SOX11 (1:1,000) (HPA000536, Sigma-Aldrich, Lot # BB107024) and Histone H3 (1:75,000) (ab4729, Abcam, Cambridge, UK, Lot # GR167613-1). Horseradish peroxidase conjugated anti-rabbit (1:2,000) (P0217, Lot # 00069121) was used as a secondary antibody (Agilent Technologies, Santa Clara, CA, USA). Chemiluminescence reaction by Amersham ECL reagent was detected with ChemiDoc^TM XRS+using Image Lab^TM Software (Bio-Rad).

Isolated proteins were resolved on 10% SDS-PAGE gels, transferred to a nitrocellulose membrane by semidry transfer (Trans-Blot Turbo^TM Transfer System, BioRad, Hercules, United States), and analysed by immunoblotting using standard procedures, as in Novačić, et al. [45 (link)]. Blots were developed with Clarity Western ECL substrates (Biorad, Hercules, United States) and visualised with a C-DiGit Blot scanner (LI-COR Biosciences, Lincoln, United States).
For Ykl162c-a experiments, strains were grown to the exponential (OD₆₀₀ 2) phase in the YPD medium, and total proteins extracted as in Kushnirov [46 (link)]. Myc-tagged Ykl162c-a was probed with anti-c-Myc (1:1,000 dilution; 9E10, Santa Cruz Biotechnology, Dallas, United States) as a primary and mouse IgG κ-binding protein horseradish peroxidase (1:50,000 dilution; Santa Cruz Biotechnology, Dallas, United States) as a secondary antibody. For Hsp150-β-lactamase experiments, isolated cell wall proteins were probed with anti-HA peroxidase-conjugated antibody (1:1,250 dilution; 11,667,475,001, Roche, Basel, Switzerland).

The protein concentration of all samples was quantified using the Bradford protein assay using BSA as a standard. 25 µg samples were loaded into precast 10% BIO-RAD Mini-PROTEAN® TGX Stain-Free^TM SDS-polyacrylamide gel (Bio-RAD Laboratories), then electrophoretically transferred to 0.2 µm nitrocellulose membrane (Trans-Blot® TurboTM Transfer Pack, Bio-RAD Laboratories) using the Bio-RAD Trans-Blot® TurboTM Transfer System. Blots were stripped between each re-probing step using stripping buffer, consisting of (in mM): Tris HCl pH 6.8: 62.5, 2-mercaptoethanol: 100, 2% SDS, 30 min at 50 °C). Quantitative densitometry was performed on images captured using a Chemidoc imaging system (Bio-rad), and ImageJ software. Data are presented as optical density of protein expression, normalized to loading control detected on the same blot.

Extracted islets were solubilized with 30 μL PBS buffer supplemented with NEM and vortexed. Subsequently, 10 μL of SDS sample buffer without reducing agent was added. Samples were boiled for 5 min at 90 °C and separated by 12% Mini-PROTEAN-TGX stain-Free^TM protein gels, and then transferred onto a Tnas-Blot-Turbo^TM Mini PVDF membrane with a Bio-Rad Trans-Blot Turbo^TM transfer system. Membrane was blocked in Tween-Tris-buffered saline with 5% (w/v) nonfat dry milk (Roth) for 1 hour and then incubated with the primary antibody A133 in Tween-Tris-buffered saline with a ratio 1:1000 overnight at 4 °C. Reactivity was analyzed with antirabbit peroxidase-conjugated secondary antibody (Serva) and chemiluminescence detection (Bio-Rad).

Samples of the recombinant proteins or bacterial extract culture before and after IPTG induction were analyzed by SDS-PAGE 12.5% under reducing conditions (2.5% dithiothreitol). After electrophoresis, proteins were stained with Coomassie Blue R-250 or transferred onto nitrocellulose membranes using the Trans-Blot^® turbo^TM transfer system (BioRad^®, Hercules, CA, USA) following the manufacturer’s recommendations. After transfer, the nitrocellulose membranes were stained with Ponceau S (Merck Millipore Corporation, Darmstadt, Germany) at 1:20 dilution to verify the transfer efficiency. To remove the dye, the membranes were washed with TBS-Tween (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) until complete removal. The membranes were incubated for 2 h with rabbit polyclonal anti-LgRec1 antibody at 1:8000 dilution. Membranes were washed with TBS-Tween and immunoreactive proteins were detected using 1:1000 diluted peroxidase labeled anti-rabbit IgG (Sigma Life Science, St. Louis, MO, USA), and the blot revealed with 4-chloro-1-naphthol. To infer the molecular mass, the following standards were used: Full-range Amersham Rainbow Marker (GE Healthcare, Little Chalfont, UK) and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA).

hMADS cell lysates were obtained by using lysis buffer containing 50 mM Tris–HCl (pH 7.4), 1% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonylfluoride, and 50 mg/ml aprotinin. Samples were centrifuged, and protein concentrations were determined by the Bradford Protein Assay (Bio-Rad Laboratories, Segrate, Italy). Proteins were separated by SDS-PAGE then transferred to a nitrocellulose membrane using the Trans-Blot Turbo^TM Transfer system (Bio-Rad). To check loading and transfer efficiency, membranes were visualized with Ponceau S solution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then blocked for 1 h at room temperature (RT) in TBS-Tween-20 (50 mM Tris-HCL [pH 7.6], 200 mM NaCl and 0.1% Tween-20) containing 5% non-fat dried milk and subsequently incubated overnight at 4°C with the primary antibody (Table 1B). After washing in TBS-Tween-20 and incubation with the secondary antibody for 1 h at RT (Table 1C), bands were visualized with the Chemidoc Imaging system using the Clarity™ Western ECL chemiluminescent substrate (all from Bio-Rad). Quantitation of immunoreactive bands was performed using the Bio-Rad Image Lab software. Where appropriate, membranes were stripped, washed and re-probed for total protein content.