10uL of each sample from yesterday (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. Gels were run @ 150V for 45mins.
Gels were 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.
Gel #1 - Decorin samples
Lane #1 - Ladder
Lane #2 - Positive control lysate
Lane #3 - Binding solution
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
Gel #2 - FST samples
Lane #1 - Binding solution
Lane #2 - Wash
Lane #3 - Ladder
Lane #4 - Positive control lysate
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
Gel #3 - LAP samples
Lane #1 - Ladder
Lane #2 - Positive control lysate
Lane #3 - Elution fraction #1
Lane #4 - Elution fraction #2
Lane #5 - Elution fraction #3
Lane #6 - Elution fraction #4
Lane #7 - Elution fraction #5
Lane #8 - Elution fraction #6
Lane #9 - Elution fraction #7
Lane #10 - Elution fraction #8
Lane #11 - Binding solution
Lane #12 - Wash
Gel #4 - Telethonin samples
Lane #1 - Binding solution
Lane #2 - Wash
Lane #3 - Ladder
Lane #4 - Positive control lysate
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
Results:
Gels all have protein, but no elution fractions exhibit just a single band. Additionally, most of the samples across the different genes show similar banding patterns.
Primary Ab (anti-6x His) was added to membrane at a 1:15,000 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:
NONE! All four membranes are completely blank. Not even a signal from the positive control lysate. Infuriating! Why? Transfer was performed in the correct orientation (this was confirmed by the presence of the ladder dye on the membranes post-transfer). The same Ab stocks and developer stock were used as a couple of weeks ago, so these shouldn’t be a concern. Ab was definitely added to all the samples, so that’s ruled out. Ugh.