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Blocking buffer coats the regions on the blot where there is no protein. It can also be used to dilute antigens, antibodies, and other proteins. Blocking buffer enhances sensitivity by:
LI-COR offers four varieties of InterceptTM Blocking Buffer: Intercept Blocking Buffer in PBS and TBS, and Intercept Protein Free Blocking Buffer in PBS and TBS. The choice between the two options depends on the experiments being performed. The Intercept Blocking Buffer is optimized for NIR fluorescent assays (NIR Western blot, On-cell Western, etc.). The buffer does not contain azides and can be used for chemiluminescent assays as well. Intercept™ Blocking Buffer is available in a variety of sizes.
Intercept Blocking Buffer (PBS, TBS) is available in 500 mL bottles and it can be bought in bulk in groups of 3, 10, 25, or 50.
Intercept Protein-Free Blocking Buffer (PBS, TBS) is available in 500 mL bottles and it can be bought in bulk in groups of 3 or 10.
Western blot protocol demonstrating the comparison of Intercept Blocking Buffer to Odyssey Blocking Buffer.
Casein Blocking Buffer is available in 100 mL and 500 mL bottles.
Advantages of Intercept Blocking Buffers
Researchers sometimes use non-fat milk or Bovine Serum Albumin (BSA) because these are low-cost options. However, their results are sub-optimal because of background illumination and they do not lead to reproducible or robust data.
Caution Against Using Powdered Milk as a Blocking Buffer
Many researchers use powdered milk as a blocking buffer, but this often times is not an ideal blocking buffer. There are several situations where milk should not be used:
Intercept T20 Antibody Diluents for use with Odyssey® Imaging Systems, are optimized for use with IRDye® Infrared Dye Reagents and other near-infrared fluorophores.
Antibody diluents are used for the dilution of primary antibodies to the working concentration required for your experiments.
Antibody diluents improve binding affinity of your primary antibody to your antigen. This reduces background due to non-specific binding.
Secondary antibodies are used in a Western blot to bind specifically to the primary antibody that is bound to the target. The secondary antibody is labeled with a detection molecule, such as HRP for chemiluminescent detection or a fluorescent dye, such as IRDye 800CW, for NIR detection.
Near infrared detection offers a several advantages over chemiluminescent detection:
Cross-adsorption is a purification process where antibodies that may react with a species other than the target are removed from the raw secondary antibody. Choosing high quality secondary antibodies is crucial in order to obtain the best Western blot results. IRDye secondary antibodies are highly cross-adsorbed, which will ensure the best possible two color data.
LI-COR provides several antibody options for infrared and visible detection.
Infrared antibodies: IRDye 800CW, IRDye 680RD, IRDye 680LT
Visible antibodies: IRDye 650, VRDye 549, VRDye490
IRDye secondary antibodies from result in cleaner blots because they are highly cross-absorbed.
Chemiluminescent detection is an enzymatic reaction between luminol, peroxide and HRP. This enzymatic reaction produces light and can be captured using film or a digital imager.
WesternSure reagents are designed and optimized for chemiluminescent research when using film, C-DiGit, Odyssey Fc imaging systems or even other CCD imaging systems. There are several reagents available in this family including WesternSure PREMIUM chemiluminescence substrate, WesternSure HRP-labeled secondary antibodies, WesternSure Pen, WesternSure Pre-stained protein ladder, and WesternSure ECL Stripping Buffer.
Available in two configurations: Goat anti-mouse (minimal cross reactivity with human immunoglobulins) and Goat anti-rabbit (minimal cross reactivity with human and mouse immunoglobulins).
Compatible with other chemiluminescent substrates, but optimized for use with WesternSure PREMIUM substrate.
WesternSure ECL Stripping Buffer is effective for reprobing with different antibodies
A low-background membrane is essential for infrared Western blot success. Background can be attributed to membrane autofluorescence or to detection of antibody non-specifically binding to the membrane. LI-COR have a variety of membranes to suit your western blot needs.
LI-COR offers high-quality, prescreened Immobilon®-FL PVDF, without lot-to-lot variability, packaged in affordable kits with the Intercept™ Blocking Buffer (PBS or TBS), or with 4X Protein Sample Loading Buffer. LI-COR also offers nitrocellulose membranes in both sheets and rolls.
LI-COR have an extensive range of reagents and consumable products. Here's a list of other reagents they have to offer.