Examples Of Specific Protocols; Sds-Protein Blotting - BIO RAD Trans-Blot SD Instruction Manual

Note: Some pH electrodes will not perform a proper measurement for the pH of Tris
buffers. If the pH of the buffer is not correct, check the electrode to be sure it is designed
to function with Tris buffers. If the pH electrode works properly with Tris buffers, and the
pH is below 9.0, remake the buffer.
2. SDS may be added to Buffer 1 to increase protein elution from the gel:
48 mM Tris, 39 mM glycine, (20% methanol), 1.3 mM SDS (0.0375%), pH 9.2
Dissolve 5.82 g Tris and 2.93 g glycine, and 0.0375 g SDS or 3.75 ml of 10% SDS in dd
H O (add 200 ml of methanol); adjust the volume to 1 liter with dd H
2
DO NOT ADD ACID OR BASE TO ADJUST pH.
3. Towbin transfer buffer for SDS-proteins using nitrocellulose (with methanol) or Zeta-
Probe membrane (without methanol):
25 mM Tris, 192 mM glycine (20% methanol), pH 8.3
Dissolve 3.03 g Tris and 14.4 g glycine in dd H
volume to 1 liter with dd H
DO NOT ADD ACID OR BASE TO ADJUST pH.
4. Dunn carbonate transfer buffer for SDS-proteins using nitrocellulose (with methanol) or
Zeta-Probe membrane (without methanol):
10 mM NaCHO
Dissolve 0.84 g NaHCO
methanol); adjust volume to 1 liter with dd H
DO NOT ADD ACID OR BASE TO ADJUST pH.
5. DNA transfer buffer for use with Zeta-Probe membrane:
5x TBE stock solution (0.5 M Tris, 0.5 M boric acid, 10 mM EDTA in dd H
volume to 1 liter with dd H
DO NOT ADD ACID OR BASE TO ADJUST pH.
6. 5x dye buffer (20% Ficoll, 20 mM EDTA, 1% SDS, 0.2% bromophenol blue)
Section 6

Examples of Specific Protocols

Note: In order to determine the optimum conditions for a particular sample, a time course
of transfer should be performed. Since many factors affect transfer, e.g., molecular weight,
pI, porosity of the gel, it may not be necessary to transfer for the full time or to use high
field intensity transfer conditions. Final transfer conditions for any protein should be
determined empirically.

6.1 SDS-Protein Blotting

Standard Blot to Nitrocellulose
1. Equilibrate the gel in 500 ml of Towbin buffer (Section 5) for 15 minutes.
2. Pre-chill buffer prior to transfer.
3. Assemble the sandwich as described in Section 4.2.
4. Refer to Section 4.2, step 9 for transfer conditions with either large or small gels.
7
O.
2
, 3 mM Na CO (20% methanol), pH 9.9
3 2 3
and 0.318 g Na
3
O. Dilute to 0.5x TBE with dd H
2
11
O (add 200 ml of methanol); adjust
2
17
CO (anhydrous) in dd H
2 3
O.
2
18
O for the working solution.
2
O.
2
O (add 200 ml of
2
O; adjust
2