No 1 filter paper

Sample extraction was carried out as described by Abubakar and Haque (2020) (link) with some modifications. Briefly, powdered plant samples about 150 g were macerated for 72 h with 1.5 L of distilled water or 95% ethanol solvent at the room temperature. After 72 h of soaking, the extracts were filtered through Whatman No.1 filter paper. The filtrates were then concentrated to dryness using a rotary evaporator at 40°C. The crude extracts were then stored at 4°C until further use.

Quantifoil or C-flat holey carbon grids (R2/1, 300 mesh) were glow-discharged using ELMO system (Cordouan) at 0.3–0.35 mBar and current of 10–15 mA for 60 s. To prepare graphene-oxide-coated grids, the aqueous dispersion of graphene oxide (GOgraphene; William Blythe Ltd) was diluted in double-distilled water (ddH₂O) to a final concentration of 1.3 mg/ml, followed by sonication in Elmasonic S 30 (H) for 120 s in a cold room and spun down at 300 g for ~2 min. C-flat holey carbon grids (R2/1, 300 mesh) were glow-discharged as described above, and 4 μl of GO solution was applied to the grids, followed by one minute incubation; subsequently, the GO solution was removed by blotting briefly with Whatman No.1 filter paper and washed by applying 20 μl ddH₂O onto the graphene-oxide-coated side twice and once on the back side of the grid with blotting steps in between.
A volume of 5 μL of TnpA^WT-IR100 mix (protein concentration of 0.06 mg/ml) was applied to a glow-discharged Quantifoil holey carbon grid (R2/1, 300 mesh), blotted from back side for 3 s at 70–90% relative humidity and plunge-frozen in liquid ethane using a Cryoplunge 3 System (Gatan). TnpA^S911R complexes with DNA substrates (5 μl) were applied to GO-coated C-flat holey carbon grids (R2/1, 300 mesh) at protein concentration of 0.18 mg/ml, blotted and plunge-frozen as described above.

A voucher of the plant is deposited in the Universidade Estadual de Maringá
herbarium with the code HUEM 24319. The fresh stem bark of S.
dulcis were washed with water immediately after collection in the
plant nursery of Universidade do Oeste Paulista, Presidente Prudente, Brazil
(UNOESTE). Then, the bark was chopped into small pieces, air dried at room
temperature for about 10 days, and turned into powder (1 kg), which was infused
in 6 L of pure ethanol for 7 days at room temperature (23 ºC ± 5). After 7 days,
the extract was filtered through cotton plugs and then through a Whatman No. 1
filter paper. The extract was concentrated under reduced pressure below 50 ºC
through rotatory vacuum evaporator (RE200 Sterling, UK). The concentrated
extract was stored at 4 ºC.

ERD acquisition was assessed according to previous studies with minor adaptations [35 (link), 36 (link), 38 (link)]. At distinct embryogenesis time points, replicates of 40 to 50 synchronous eggs were placed on a Whatman No. 1 filter paper and air-dried for 15 min. Next, shrunken and intact eggs were counted using a stereomicroscope, and ERD evaluated. Results were obtained after three independent experiments, in which the strains were simultaneously tested.

Dried plant aerial parts (leaves) were pulverized. Each 15 g of ground sample was placed into a separate Erlenmeyer flask. Then, 100 mL of ethanol (100%) was added, and the samples were incubated in a water bath at 55 °C for 6 h. Separation of the extraction mixture from the residue was achieved by filtration through Whatman No. 1 filter paper. Each plant residue was re-extracted in triplicate with ethanol. After filtration, the two portions were mixed. The residual solvent in the ethanolic extracts were removed under reduced pressure at 48–49 °C using a rotary evaporator (Rotavapor IKA VB 10, Germany). Water in the extracts was lyophilized using a freeze dryer (Thermo Savant Modulyo D, USA) for 8 h at − 50 °C and 0.040 mbar. The yields of these fractions were 20.16% (Ocimum basilicum) and 15.78% (Thymus algeriensis).

Fresh young alfalfa leaves were harvested, dried, and ground at room temperature. Approximately 2 g of the powdered sample was boiled in 50 mL of methanol-distilled water (4:1) for 6 h. To remove debris, all the sample solutions were filtered through Whatman No. 1 filter paper. The extracted solvent (80% methanol) was evaporated at 40 °C in a vacuum rotary evaporator. The extract was dissolved in ether to precipitate the saponins, at room temperature. The obtained solution was centrifuged at 4 °C for 20 min. Purification of saponins in the precipitate sample was performed as previously described [86 (link)]. Briefly, 10 g of crude saponin samples were suspended in 100 mL CHCl. Thin layer chromatography (TLC) was performed to visualize the patterns of unknown compounds. After repeated chromatography, compound identification in the samples was performed using NMR analysis.