This protocol is based off of SB and SK’s notes and Bio-Rad documents (Bulletin 6376, 2895 and M1703930). This WB protocol is optimized for a primary anti-FLAG antibody which is used for general conformation of expression. Before starting this protocol, do know that this process will take between one-half and a full workday in the lab. There are numerous, one hour plus break points. If necessary, blocking/antibody incubation can be done overnight at 4° C (but do not rock while doing this!). Most problems encountered arise during the transfer portion of the experiment. Manipulating transfer times, temperature, voltage, etc. all will require inputs of sample, so before starting a WB do try to have at least 5x excess sample for multiple repeats. For example, if the SDS-PAGE gel well holds 15uL, try to keep 75uL of sample just in case you need to repeat the WB. Also note all methanol or methanol containing buffer needs to be disposed of in hazardous waste containers. When the container is full, fill out a chemical waste pickup form. DO NOT DISPOSE OF METHANOL IN THE SINK. Methanol is a flammable, hazardous waste and should be treated as such.

Buffers Required

1. 1L Running Buffer (use 10X)
2. 5L Transfer Buffer (Running buffer + 20% Methanol) (chilled in -20°C for one hour prior to use)
3. 0.5L TBST (20mM Tris pH 7.5, 150mM NaCl, 0.1% Tween-20)
4. 50mL Blocking Buffer (5% milk or 3% BSA in TBST)

Materials Required

1. Pencil and Ruler
2. Razer blade
3. 1x Immunoblot PVDF (7.5cm x 10cm)
4. 2x Mini Transblot Filter Paper
5. 1x Ice Block from -20C
6. 4x Western Blot pads
7. 3x Gel boxes
8. 1 Transfer Pipette
9. Saran Wrap
10. Development Solution