western blot quantification imagej and ccd imager

WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER MANUAL
WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER SOFTWARE
WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER PLUS

WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER SOFTWARE

The developed membrane can be imaged using a CCD camera that enables the bands’ intensity to be analyzed using software such as Image J. However, this may lead to unacceptable background signal. Poorly expressed proteins may require prolonged incubations with the substrate of (up to) several hours or even overnight to produce a signal with suitable intensity. Less target protein results in fewer antibody complexes binding and consequently less conjugate. The speed of signal development is dependent on the abundance of the reporter enzyme to catalyze the reaction, which is governed by the abundance of the target protein and the primary antibody bound to it. Color development is monitored visually until it reaches satisfactory strength, following which the reaction may be stopped by washing excess substrate from the membrane or by applying a stop buffer to halt the reaction. The sensitivity of the substrate and the length of incubation determines the intensity of colored precipitate deposited onto the membrane.

Modern formulations offer substrates in stabilized peroxide, which can provide more consistent results in a ready-to-use application. Traditionally, hydrogen peroxide is added to the buffer prior to use, giving the substrates a shelf-life of only a few hours. It is necessary to add Peroxide to the substrate when using HRP conjugates. Much higher sensitivities can be achieved using NBT/BCIP for AP and 4CN/DAB for HRP as combination substrates. Each substrate has a different affinity for its respective enzyme. Colorimetric Substrates for Horseradish PeroxidaseĬommonly used substrates for HRP include 3,3′, 5,5′-tetramethylbenzidine (TMB), 3,3-diaminobenzidine (DAB) and DAB in combination with 4-chloro-1-naphthol (4CN), which appear as dark blue, brown and black precipitates, respectively. Nitro blue tetrazolium (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) are two AP substrates used in combination with colorimetric detection to give a blue-purple precipitate. The R² value of the plotted data for the entire range of total protein signal was determined to be 0.9990, whereas the R² values for β-actin, GAPDH, and α-tubulin were 0.8851, 0.9438, and 0.8332, respectively.Colorimetric Substrates for Alkaline Phosphatase The iBright software was used to quantitate the total protein signal in the lanes. The blot was imaged using the Invitrogen™ iBright™ FL1500 Imaging System.

WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER PLUS

138000), and Invitrogen™ Goat Anti–Mouse IgG Alexa Fluor™ Plus 680 secondary antibody (Cat. The membranes were then washed 3 times for 2 minutes with 20 mL of ultrapure water on a rotating platform, followed by addition of Invitrogen™ primary antibodies against β-actin (Cat. The PVDF membranes were washed twice for 2 min with 20 mL of ultrapure water on a rotating platform, whereupon they were labeled with 10 mL of a working solution of No-Stain Protein Labeling Reagent on a rotating platform for 10 minutes. Proteins from the gels were transferred onto mini PVDF membranes using the Invitrogen™ iBlot™ 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 minutes). Invitrogen™ Bolt™ 4-12% Bis-Tris Plus gels were loaded with HeLa lysate ranging from 10 to 50 µg and electrophoresed using MES running buffer. Total protein normalization using the No-Stain Protein Labeling Reagent.

Image view window: display selected image in the window for user view and interaction.

Color option: select color or grayscale image view.

Split screen: select one-window or two-window image view.

Export: edit image information and export current images to destinations of choice.

Analyze: initiate analysis workflow using current images.

Live view: go back to live-streaming sample view.

More options: open image adjustment options.

Edit channels: open multiple channel options to review and edit each channel.Capture: acquire images using exposure time set by user or instrument.

WESTERN BLOT QUANTIFICATION IMAGEJ AND CCD IMAGER MANUAL

Exposure dial: select or turn the dial to set manual exposure time or fine-tune exposure time.Drawer: open or close the sample drawer.Settings: access general instrument settings, configurations, and service tools.Camera lock: maintain camera settings across multiple samples, locks exposure time, zoom, and transillumination settings while disabling mechanical auto-rotation.Mode selection: dropdown menu to select imaging modes (chemiluminescent blot, fluorescent blot, nucleic acid gel, protein gel, and universal mode).Gallery: access previously captured images.Sign in: create a new user account of sign in to an existing account.

Western blot quantification imagej and ccd imager