10uL of each sample from 20081112 (binding supe, washes and elution fractions) were mixed with 2x sample reducing buffer and boiled 5 mins. Samples were loaded onto Pierce 4-20% Tris-Glycine SDS/PAGE gels. 10uL of SeeBlue ladder was loaded. 5uL of positive control lysate was loaded. Gel was run @ 150V for 45mins.
Gel was transferred to nitrocellulose membrane (equilibrated in tris-glycine transfer buffer for 5mins) @15V for 15mins. Membrane washed briefly in 1x TBS-T, then blocked for 1hr. Gel was stained for 1hr in Coomassie stain and then destained w/ 10% acetic acid until bands were clearly visible.
Lane #1 - Ladder
Lane #2 - Positive control lysate
Land #3 - Binding supe
Lane #4 - Wash
Lane #5 - Elution fraction #1
Lane #6 - Elution fraction #2
Lane #7 - Elution fraction #3
Lane #8 - Elution fraction #4
Lane #9 - Elution fraction #5
Lane #10 - Elution fraction #6
Lane #11 - Elution fraction #7
Lane #12 - Elution fraction #8
Primary Ab (anti-6x His) was added to membrane at a 1:10,000 dilution. This incubated for 1hr. Used Ab solution was stored @ 4C.
Membrane was washed with 1x TBS-T and fresh blocking solution was added per the protocol.
Secondary Ab (DAR HRP) was added to membrane @ 1:15,000 dilution and incubated for 30mins.
Membrane was washed with 1x TBS-T per the protocol.
Blots were developed using Millipore Immobilon chemiluminescent regent and imaged.
Results:
Western is totally blank. Again. Absolutely no signal at all detected. Not even a smudge/blob/dot/forceps mark/crease. Nothing.