Western Blot Materials
Semi-dry Transfer Apparatus	Biorad, Cat# 170-3940, or equivalent
Power Supply	0 - 100 VDC (adj. current to 1 Amp)
Immobilon-P transfer membrane	0.45 µm pore size; cut to same size as gel (Millipore, Cat# IPVH304-FO, or equivalent)
Filter Paper	Whatman 3MM or equivalent, cut to same size as gel
Wetting Solution	100% Methanol
Anode Buffer I	300 mM Tris, 20% methanol, pH 10.4
Anode Buffer II	25 mM Tris, 20% methanol, pH 10.4
Cathode Buffer	25 mM Tris, 20% methanol, 40 mM 6-aminohexanoic acid, pH not adjusted
Additional Tools	Forceps, clean plastic test tube, gloves, razor blade

R&D Systems’ QC laboratories use these western blotting and immunostaining protocols to show that our polyclonal and monoclonal antibodies are specific for the proteins they were raised against and to determine the sensitivity of the antibody for its antigen. Protein samples are prepared with SDS and run non-reduced and reduced on an appropriate SDS-PAGE gel. The proteins are transferred to a PVDF membrane using a semi-dry transfer apparatus.

Some antibodies require different Western blotting conditions, please see the package insert for specific conditions.

1. Prepare reduced and non-reduced samples/controls and separate proteins using a SDS-PAGE gel. Use appropriate percentage SDS-PAGE gel for protein of interest. Typically 12% acrylamide gels are used for high molecular weight (MW) proteins (>50 kDa), 15% gels for mid range MW proteins (15 - 50 kDa) and 20% gels for low MW proteins (<15 kDa). Use pre-stained MW standards as an indication of a successful transfer.
2. Remove stacking gel and sides of running gel beyond sample wells with a razor blade. Notch bottom right-hand corner of gel for orientation. Note: Equilibration of running gel in Cathode Buffer is generally not required but may improve results if bands appear diffuse.
3. Prepare transfer membrane by soaking in Wetting Solution for a few seconds. Equilibrate membrane in Anode Buffer II for 5 minutes.
4. Wet two pieces of filter paper in Anode Buffer I and place on anode plate of blotter. Avoid trapping air between electrode and filter paper by laying filter paper on electrodes at an oblique angle.
5. Wet one piece of filter paper in Anode Buffer II and place on top of filter papers previously placed on electrode.
6. Remove transfer membrane from Anode Buffer II and place on top of filter paper stack.
7. Place gel on top of transfer membrane taking care not to trap air bubbles between gel and membrane.
8. Wet three pieces of filter paper in Cathode Buffer and place on top of gel. Use a clean plastic test tube to roll out air bubbles.
9. Place cathode plate of blotter on top of transfer stack (see Figure 1).
10. Connect high voltage cords to power supply. Apply a constant current of 1.9 - 2.5 mA per cm2 of gel area for 30 - 60 minutes. Transfer time depends upon proteins being transferred.
11. After transfer is complete, turn off power supply and remove cathode plate of blotter. Remove transfer membrane and cut lower right corner of membrane to mark orientation of gel.
12. If immunostaining is to be performed immediately, rinse the membrane in distilled water and proceed to the immunostaining portion of protocol. If staining is to be performed at a later time, place membrane on a paper towel and allow to dry. Store membrane in a dry container at 4 °C.

Western Blot Protocol for Cell Lysates

This western blotting protocol is intended to provide additional detail for the use of R&D Systems antibodies in western blotting. Please consult the product insert for the appropriate concentration of primary antibody and the composition of Wash Buffer, Blocking Solution, and Blotting Buffer.

To prepare total cell lysates, add enough PBS to cell pellet to make the final concentration 3 x 106 cells/ml. This is a convenient cell density for many cell lines, but adjustments may be necessary for cell types that differ substantially in size and protein content. Prepare cell extracts in appropriate non-reducing or reducing sample buffer as indicated on the product insert. In some cases reducing agents can destroy the epitope that is recognized by a monoclonal detection antibody. Mix cell suspension with an equal volume of non-reducing 2X SDS gel sample buffer (6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, and bromophenyl blue) or reducing 2X SDS gel sample buffer (non-reducing buffer plus 20 mM dithithreitol). Sonicate cells to fragment DNA using 8-10 bursts of 2-3 seconds each.

1. Load cell extracts and separate proteins on an SDS polyacrylamide gel.
   
2. Transfer the electrophoresed proteins onto an Immobilon P membrane (Millipore) and incubate the membrane for 1 hour at room temperature or overnight at 2-8° C in Blocking Solution.
3. Wash the membrane at room temperature for 30 minutes with 3 or more changes of Wash Buffer.
4. Incubate the membrane for 2 hours at room temperature or overnight at 2-8° C in Blotting Buffer containing primary antibody.
5. Wash the membrane at room temperature for 30 minutes with 3 or more changes of Wash Buffer.
6. Incubate the membrane at room temperature for 1 hour in Blotting Buffer containing an appropriate secondary reagent such as goat anti-mouse-HRP at 1:4000 .
7. Wash the membrane at room temperature for 30 minutes with 3 or more changes of Wash Buffer.
8. Detect with chemiluminescence reagents.
9. Optimal dilutions should be determined by each laboratory for each application.