Tag Archives: chemiluminescent

SDS/PAGE/Western – anti-HSP70 Ab Re-test

Another attempt to determine appropriate amounts of anti-HSP70 Ab (ABR cat# MA3-006)and/or protein needed for better detection of HSP70 in Gigas protein samples. Gigas gill protein extracts from 20080617 (both control and Vibrio exposure samples) were each pooled. The two samples were mixed with an equal volume of 2x sample reducing buffer. 100uL of hemolymph were extracted from Gigas muscles and mixed with an equal volume of 2x sample reducing buffer. Samples were boiled for 5mins. and loaded onto a Pierce 4-20% tris-hepes gel. Also loaded 10uL of SeeBlue ladder. Ran gel @ 150V for 45mins. Samples were transferred to nitrocellulose 20V for 30mins. Well locations were marked on the membrane with a pencil. Gel was stained with Coomassie stain for 30 mins and then destained with 10% acetic acid until bands were clearly visible.

external image 20081216.JPG

Lane 1 – Ladder
Lane 2 – Control (20ug)
Lane 3 – VE (20ug)
Lane 4 – Hemos (40uL)
Lane 5 – Control (10ug)
Lane 6 – VE (10ug)
Lane 7 – Hemos (20uL)

Lane 8 – Control (20ug)
Lane 9 – VE (20ug)
Lane 10 – Hemos (40uL)
Lane 11 – Control (10ug)
Lane 12 – VE (10ug)

Membrane was cut in two (between lanes 7 & 8) for probing with two different primary Ab concentrations: 1:5000 (per the manufacturer’s suggestion) and 1:2500. Membranes were incubated 1hr. in 15 mL of blocking solution. Primary Ab was added at the two above mentioned concentrations to the respective membrane and incubated 1hr. Membranes were washed with 1x TBS-T 3 x 10mins. Secondary Ab (DAM-HRP) was added with blocking solution to the membranes at a 1:2500 dilution and incubated 30mins. Membranes were washed with 1x TBS-T 3 x 10mins. Membranes were developed with Millipore Immobilon Western Chemiluminescent reagent and imaged together.

Results:

No image of any sort! Not even the pencil marks were visible. Just a blank screen. I’m starting to suspect that something is wrong with the imaging system or something. This is basically a repeat of the Western on 20081210 which worked. Now I’m not sure what to do at all. This blows.

SDS/PAGE/Western – Attempt to fix/identify problem(s) with Westerns

Will use two different antibodies and two different development methods: chromogenic (Invitrogen Western Breeze) and chemiluminescent.

Loaded four sets of MSTN1b active form protein (in 1x TBS) of 15uL protein + 15uL reducing sample buffer (after boiling for 5mins.) onto a Pierce 4-20% tris-hepes gel. Also loaded four sets of 10uL SeeBlue ladder, along with 5uL of positive control lysate. These were each loaded into the same lane. Ran gel @ 150V for 45mins. Samples were transferred to nitrocellulose 15V for 15mins. Gel was stained with Coomassie stain for 45 mins and then destained with 10% acetic acid until bands were clearly visible.

Samples are loaded as: Ladder, MSTN1b, blank. Repeat across gel.

Membrane was cut into four pieces: two for standard Western blotting and two for the colorimetric development. These were blocked in 15mL of blocking solution (standard) or 10mL of blocking solution (chromogenic) for 30mins. Primary antibodies used were anti-c-Myc (mouse) and anti-His (rabbit). For the standard Western, they were used at a 1:5000 dilution. For the colorimetric, they were used at a 1:1000 dilution (per the antibody info sheet). Membranes were incubated with primary antibodies for 1 hr. Membranes were washed per the protocol. Secondary antibodies (GAR AP for chromogenic; DAR HRP & GAM HRP for chemiluminescent) were added. 1:3000 dilution for chromogenic and 1:5000 dilution for chemiluminescent. Incubated for 30 mins. Membranes were washed per the protocol.

Chromogenic: Added substrate to membranes and incubated for ~5mins. until band was very prominent.

Results:

Prominent band is MSTN1b. Smearing is commonly seen with this recombinant protein. Positive control lysate (12-tag marker) was not detected.

 

 

Chemiluminescent: Added 4mL total of Millipore Immobilon chemiluminescent reagent to both membranes. 10 min. expsoure.

Results:

Membrane on the left only shows detection of MSTN1b and not the positive control lysate. The membrane on the right shows both MSTN and the positive control lysate.

SDS/PAGE/Western – anti-HSP70 Ab test CONTINUED (from yesterday)

Primary Ab (anti-HSP70) solution was saved and stored in the 4C. The membrane was washed 3x 10 mins. w/1x TBS-T. 15mL blocking solution was added to membrane and incubated at RT for 10mins. Secondary Ab (DAM HRP) was added at a 1:2500 dilution and inubated at RT for 30 mins. Secondary Ab solution was discarded and membrane was washed 3x 10 mins. w/ 1x TBS-T and then developed with Millipore Immobilon Chemiluminescent reagent.

Results: There is a small “blob” towards the upper left of the membrane that is likely HSP70, based on the expected molecular weight. That band corresponds to the control gigas gill pool sample (see yesterday’s SDS/PAGE).

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.