Coomassie brilliant blue R

Wound healing was performed using a 96 well floating-pin transfer device with a pin diameter of 1.58 mm coming to a flat point at the tip with a diameter of 0.4 mm (VP Scientific VP-408FH). Foam backing was inserted between the plates of the pin array to provide a resistive stop and the external guide pins were bent to allow greater movement in the z-axis. The pin array was placed in the top corner of a well, pushed down into the plate to engage all pins, and then pulled toward the user. This was repeated in the three neighboring wells to cover all 384. Plates were returned to the tissue culture incubator for 7 or 24 hours before fixation.
Cells were fixed after removal of the media with a wand aspirator (VP scientific VP-186L) used along with the Labsystems Multidrop for all liquid handling. Fixation solution (100 mM K-Pipes pH 6.8, 10 mM EGTA, 1 mM MgCl₂, 0.2% Triton X-100, 3.7% Formaldehyde) was added as 30 μl and incubated for 15 min. Wells were aspirated and washed 2× with TBS with 0.1% Triton-X 100 (TBS-Tx) and stained.
For experiments involving the automated microscope and macroscope, cells were stained in TBS-Tx with TRITC-phalloidin (Sigma P1591) 0.5 μg/ml and Hoechst (Sigma B2261) 0.1 μg/ml as 15 μl per well for 15 minutes. Wells were washed 2× with TBS-Tx and imaged. For experiments using the fluorescence plate scanner, after fixation, cells were incubated in TBS-Tx with 2% BSA (AbDil) for 30 minutes, incubated with mouse anti-actin antibody (Chemicon MAB1501) at 1:10,000 in AbDil for 45 minutes, washed 2× with TBS-Tx, incubated with secondary antibodies appropriate for the plate scanner (Molecular Probes A-21057), washed 2× with TBS-Tx and imaged. For experiments using the transmitted-light scanner, after fixation, cells were incubated with SDS-Page gel staining solution (0.25% Coomassie Brilliant Blue R-250, 50% methanol, 10% acetic acid) for 10 minutes, washed 2× with TBS and imaged.

The TspGST gene (GenBank accession no. XM_003373603) was amplified by PCR using specific primers with BamHI and Hind III restriction enzyme sites (underlined) (forward: 5′-TAT AGG ATC CAT GAC CAA CAC GTC GAA GAA AGG-3′; reverse, 5′-GCC CAA GCT TTC ATT GAC TTT CAA TAG TCA CTG G-3′). The purified PCR product was cloned into the pMD19-T vector (Takara, Dalian, China), subsequently sub-cloned into the pQE-80 L (Novagen, La Jolla, CA, USA). The recombinant plasmid carrying the TspGST gene was transformed into Escherichia coli BL21 (DE3) (Novagen), and expressed under IPTG induction. The rTspGST was purified using Ni-NTA-Sefinose resin (Sangon Biotech, Shanghai, China). The concentration of the purified rTspGST was assayed as described previously [22 (link)], and identified by SDS-PAGE analysis [23 (link)]. The gel was stained with 0.25% Coomassie brilliant blue R-250 (Sigma-Aldrich), and subsequently decolorized.

SDS-PAGE electropherograms were used to study the degradation of gluten proteins during fermentation, and the operation was referred to as the method of Lee et al. (24 (link)) with slight modifications. Gluten fermentation broth with different fermentation time intervals was taken and kept in a water bath shaker (150 r/min) for 1 h at 50°C and mixed well and then centrifuged at 8,000 × g, 4°C for 10 min to take the supernatant and set aside.
Prepare the gels according to the kit (4% concentrated gel and 15% separation gel). Add the supernatant proportionally to the sample buffer, then add the sample to the gel, and perform electrophoresis at 120 V. After completion, the gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 and scanned using Image Scanner III. A quantitative analysis of the stained protein bands was performed.

Twelve percent SDS PAGE was done for estimation of the purified GST subunits [29 (link), 42 (link)]. Isoelectrofocusing PAGE was done for estimation of the pI values [35 (link), 41 (link)]. Staining of proteins was achieved with 0.25% Coomassie Brilliant Blue R-250.