SDS/PAGE/Western – Purified (His column) FST samples from 20081112

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.

Western Blot – Purified (His column) decorin, FST, LAP & telethonin

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.