Chemidoc xrs imaging system

One milliliter of logarithmically growing yeast cells was harvested for protein extraction. Cells were immediately pelleted and resuspended into 250 μl of 0.1 N NaOH for 5 min. The NaOH was then removed by centrifugation so that the cell pellet could be resuspended in 1× Laemmli Sample Buffer. This resuspension was boiled for 5 min. Total protein concentration was determined using an RC/DC assay (BioRad 5000121). Equal amounts of protein were electrophoresed on 8% or 10% SDS-PAGE gels and transferred onto Immobilon-PVDF Transfer membranes. (Millipore IPVH00010). Immunoblot analysis was performed using standard procedures. All blots were scanned with a BioRad ChemiDoc XRS+ Imaging System. Band intensities were quantified using ImageJ 1.51 v software.
Sen1 immunoblots were performed with slight differences from the standard protocol above as described previously (44 (link)). First, samples were electrophoresed using 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad 4561086) at 140 V for 1 h transferred onto Immobilon-PVDF Membrane. Blots were blocked with 5% dried milk in TBST buffer with 0.1% Tween-20 at 23°C for 1 h, incubated with Sen1 antibody (1:2000 dilution) for 1 h and then an anti-rabbit secondary at 1:3000. Blots were scanned with a BioRad ChemiDoc XRS+ Imaging System and band intensities were quantified using ImageJ 1.51 v software.

Relative amounts of LF were quantified using previously described methods [33] . Briefly, the exotoxin-containing supernatants used in the cytotoxicity assays were quantified using the D_C Protein Assay Kit (Bio-Rad, Hercules CA), normalized to 40 µg in a final volume of 30 µL of 1x Laemmli buffer, and heated at 95°C for 4 minutes. Total protein amounts were measured by staining with MemCode Reversible Protein stain (Thermo Scientific, Rockford IL) and relative amounts quantified by densitometry using the ChemiDoc XRS Imaging system and Image lab v4.0 (Bio-Rad, Hercules CA). The membrane was blocked in a 4% Non-fat milk/TBS +0.05% Tween-20 (TBS-T) solution at 4°C overnight and then incubated for 1 hour with 1∶2500 α-LF (DD-6; BEI, Manassas, VA). After washing with TBS-T, the membranes were incubated for 1 hour with 1∶1000 goat α-mouse IgG Alkaline phosphatase. Bands were developed by incubating with Immuno BCIP/NBT substrate solution (MP Biomedicals, Solon OH) until bands were clearly visible. The amount of LF was then quantified using densitometry via the ChemiDoc XRS Imaging system and Image lab v4.0 (Bio-Rad, Hercules CA).
Relative quantitation was calculated using the following formula:

1:10 SYBR green I (Life Technologies) dilution was made in TAE buffer, then 2 μL of the SYBR dilution was added to the RT-LAMP reactions. The visual change of color (orange to yellow) can be used to identify positive amplifications. In addition, the SYBR green I PCR tubes were imaged under UV light in the Bio-Rad ChemiDoc XRS+ Imaging System as the reaction creates a fluorescent output. One half of the RT-LAMP reaction was electrophoresed along with Invitrogen Low DNA Mass Ladder on a 2% agarose gel in 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) at 90 V for 90 minutes. Gels were imaged under UV light using the Bio-Rad ChemiDoc XRS+ Imaging System. Lanes containing a laddered banding pattern were qualified as a positive amplification. Clinical samples were considered positive for RT-LAMP products if they had both a fluorescent signal by excitation of UV light and if they had a laddering pattern when run on a gel.

Pancreatic cancer P198 shGTK‐KD and shControl cells were lysed to extract proteins for protein concentration. These proteins were used for western blotting as described in the Supporting Information. The protein expression image was subsequently developed using a ChemiDoc XRS+ Imaging System.

The membrane proteins were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Western blot was used to analyze CD47 in the RBC lysate, RBCs, and bFGF-RBC/NP. The results were observed using a ChemiDoc-XRS imaging system.
The size, polydispersity index (PDI), and zeta potential of bFGF-NP and bFGF-RBC/NP were measured using a dynamic light scattering detector (Litesizer, Anton Paar, Austria). Thereafter, bFGF-RBC/NP was incubated in PBS and subsequently PBS with 10, 20, and 40% FBS. The diameter and PDI changes were monitored for 7 days to analyze the stability under physiological conditions. Subsequently, the morphology was observed under a transmission electron microscope (TEM, JEM-100CX, Japan). The encapsulation efficiency of bFGF-RBC/NP containing various concentrations of bFGF was determined.
In vitro release of bFGF was evaluated using the dynamic dialysis method. bFGF, bFGF-NP, and bFGF-RBC/NP were placed into dialysis tubes (100 kDa) and immersed in PBS at 37°C for 72 h under horizontal shaking at 100 rpm. PBS was collected and replaced with an equal volume of fresh PBS periodically. bFGF in PBS was detected using the ELISA kit.

Total protein isolated from the myocardium was separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked with 5% fat-free milk and incubated overnight at 4°C with primary antibodies including Cav-1 (1 : 1000), VEGF (1 : 1000), VEGFR2 (1 : 1000), GADPH (1 : 10000), ERK1/2 (1 : 1000), and p-ERK1/2 (1 : 2000). After washing with TBST for three times, the membranes were incubated with secondary antibodies (1 : 10000) for 2 h at room temperature. ChemiDoc™ XRS+ Imaging System was used to visualize the signals. Javas freely available NIH ImageJ software (NIH, Bethesda, MD, USA) was used to quantify the intensity of immune reactivity.