Tag Archives: Western blot

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.

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).

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.