ChemiBLOCKER

Western Blot

ELISA

PROTOCOL:

1. Make up blocking solution by mixing 1 part ChemiBLOCKER with 1 part 1X femto-TBST/PBST (TBST or PBST also work) for blocking PVDF membranes. For nitrocellulose membranes, mix 1 part ChemiBLOCKER with 3 parts 1X femto-TBST (TBST or PBST also work). Note:ChemiBLOCKER can be used at a wide variety of dilutions, some optimization may be necessary. ChemiBLOCKER is NOT recommended for phospho-protein studies as it does contain phosphorylated proteins.

2. Incubate the membrane in blocking solution (10-20 mL) for 60 minutes at room temperature with shaking in a heat-sealable plastic bag.

3. Prepare antibody dilution buffer (ADB). For PVDF membranes mix 1 part ChemiBLOCKER with 3 parts 1X femto-TBST/PBST (TBST or PBST also work). Incubate the membrane in 5-10 mL primary antibody, diluted appropriately with ADB, for 90 min at room temperature with shaking.

4. Wash the membrane 3 times in 50 mL of 1X femto-TBST (TBST or PBST also work) for 5 min each time at room temperature with shaking.

5. Incubate the membrane in 5-10 mL secondary antibody, diluted appropriately with ADB, for 90 min at room temperature with shaking.

6. Wash the membrane as in step #4, but add one extra wash (4 washes total).

7. Wash the membrane 3 times quickly (30 sec.) in 50 mL pure deionized water each time and then process the blot, using femto-LUCENT detection system (or your favorite system).

The ChemiBLOCKER is an unique membrane-blocking agent. As a non-animal protein blocker it offers an alternative to milk-based blocking agents, minimizing the risk of non-specific binding of antibodies to your membrane and lowering your background. ChemiBLOCKER may be used at dilutions as high as 20x but for most applications, a 5 to 10 fold dilution is appropriate. ChemiBLOCKER is biotin free and will not interfere with ABC detection systems. ChemiBLOCKER does contain phosphates and may not be suitable for use with some phospho-specific antibodies.

Related Products:
• BLOT-QuickBlocker A novel blocking protein which produces a semi-clear solution. This allows users the option to examine their blot membranes during the blocking procedure. 200g Catalog Number 2080
• Femto/TBST wash buffer (10X) 250mL Catalog Number 2081

Materials required:
• 1X femto/TBST (Catalog Number 2081), 1X TBST, or 1X PBST
• primary and secondary antibodies
• reagents for immunodetection

2X ChemiBLOCKER 1 L

sufficient for 100 mini gel blots

A 2X solution of non-animal proteins in 2mM TRIS, pH 8.5, 100mM NaCl, 0.001% Tween-20, and 0.08% Kathon added as a preservative.

Store kit components at 2-8°C for up to 1 year from date of purchase. Warm ChemiBLOCKER to room temperature and mix thoroughly before dispensing. To avoid possible microbial contamination, dispense all solutions aseptically. Over time, phenolic compounds may precipitate out of solution resulting in the formation of a small amount of dark brown to black particulate material at the bottom of the container. This material will not affect the performance of the blocking compound.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany