Tag Archives: Western Breeze Chromogenic (anti-mouse) Kit

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 – 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.