Tag Archives: Coomassie

SDS/PAGE, Western Blot – Test of HSP70 Ab on heat stressed shellfish for FISH441

Pacific oysters, a mussel, barnacles and a clam (sp. ?) were transferred from the holding tank to a large beaker with sea water which was placed into a 37C water bath. The shellfish were incubated in this water bath for ~3hrs. Tissues were collected from each, transferred to a 50mL conical tube and immediately placed in a dry ice/ethanol bath:

Oyster – gills, muscle and mantle

Clam – whole clam

Mussel – whole mussel

Barnacles – whole barnacles

0.02 – 0.07g of each tissue were weighed, added to a 1.5mL tube containing 0.5mL CelLytic MT + protease inhibitors and homogenized. For the barnacles, ~8 barnacles were transferred to a weigh boat and smashed with a hammer. This was then transferred to a 1.5mL tube with CelLytic MT + protease inhibitors. Tubes were spun @ max speed @ 4C for 10mins. Supe was transferred to a fresh 1.5mL snap cap tube. 15uL of each sample was transferred to a screw cap tube containing 15uL of 2X reducing sample buffer. Tubes were boiled for 5mins and then spun for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder was also loaded on the gel.

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membranse for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary HSP70 Ab was used at a 1:3000 dilution (per the Meistertzheim et al. paper).

SDS/PAGE, Western Blot – Test of new Western Breeze Kit & HSP70 Ab for FISH441

Ran varying amounts of Gigas Gill 24hr Control protein extract from 20080917. See table below:

Protein (ug) Volume of Sample (uL) Water up to 15uL (uL)
5 1.56 13.44
10 3.125 11.88
15 4.69 10.31
20 6.25 8.75
30 9.38 5.63
40 12.5 2.5
50 15.65

Volumes were adjusted to 15uL with H2O and combined with 15uL of 2X Reducing Sample Buffer in screw cap tubes. Also, 15uL of MSTN was mixed with 15uL of 2X Reducing Sample Buffer in a screw cap tube. This will serve as a positive control for the kit. Samples were boiled for 5mins. and then microfuged for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder and 10uL of the 12-tag positive control lysate were also loaded on the gel.

external image 20081231.JPG

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membrane for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

After transfer, membrane was cut into two (one with the gigas samples and the other with the MSTN and the 12-tag lysate samples) and developed separately. Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary anti-HSP70 Ab was used at a 1:5000 dilution (2uL) and the primary anti-myc tag Ab (for MSTN) was used at a 1:1000 dilution (10uL). The anti-myc blot was allowed to develop for ~1min before stopping. The anti-HSP70 blot was allowed to develop for ~1.5hrs before stopping.

external image 20081231-01.JPGexternal image 20081231-02.JPG

Results:

 

The anti-HSP70 blot showed no bands. The anti-myc blot showed MSTN1b and positive control lysate bands. This confirms that the kit works properly.

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

Test of the new anti-HSP70 Ab (ABR cat# MA3-006). Gigas gill protein extracts from 20080617 (both control and Vibrio exposure samples) were each pooled to result in 10ug protein of control and 10ug protein of VE in a volume of 10uL each. The two samples were mixed with an equal volume of 2x sample reducing buffer. O. rubescans samples were taken from Rachel’s -20C box. 15uL of each sample was mixed with 2x sample reducing buffer. All samples were boiled for 5mins and spot spun. Samples were loaded onto Pierce 4-20% Tris-Glycine SDS/PAGE gels. 10uL of SeeBlue ladder was loaded. Gel was run @ 150V for 45mins.

Gel was transferred to nitrocellulose membrane (equilibrated in tris-glycine transfer buffer for 5mins) @15V for 20mins. Membrane washed briefly in 1x TBS-T, then blocked for 1hr. in 15mL of blocking solution. Primary Ab was added at a 1:5000 dilution per the HSP70 data sheet and incubated O/N @ 4C. Gel was stained for 1hr in Coomassie stain and then destained w/ 10% acetic acid O/N.

Lane #1 – Ladder

Lane #2 – Gigas gill control pool

Lane #3 – Gigas vibrio exposure pool

Lane #4 – O. rubescans “mucus from suction” 10/24

Lane #5 – O. rubescans “UW 11/21″

Lane #6 – O. rubescans “Bucket 11/21″

Lane #7 – O. rubescans “water/mucus from bucket 10/24″

Lane #8 – O. rubescans “skin 11/21″

Results: The two oyster samples are the only two that are visible on the coomassie stained gel. I will have to talk to Rachel concerning the columes of O. rubescans proteins that she loaded on her gels, as it is NOT clear in her notebook entries. Additionally, the gel did not destain very well.

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.