rRNA removal was continued from O/N precipitation. Processed the samples according to the Ambion MICROBExpress Kit protocol and resuspended final pellets in 25uL of The RNA Storage Solution. Samples were spec’d on the NanoDrop.
Samples were stored @ -80C in Sam’s RNA Box #1.
Added 10mL TriReagent to 2 x 50mL pellets (5.63 x 10^11 total bacteria; see calcs on 20081219) from the control samples collected on 20081219. Added 10mL TriReagent to 1 x 50mL pellet (1.835 x 10^12 total bacteria; see calcs on 20081219) from the V.tubi + oyster samples collected on 20081219. Scaled rest of RNA protocol to match. Resuspended pellets in 100uL 0.1%DEPC-H2O. Samples were NanoDropped.
The V. tubi + gigas sample was eventually diluted to contain 400uL (see final reading for that sample above).
RNA samples were precipitated O/N @ -20C according to Ambion protocol.
Significantly more bacteria in the container containing autoclaved oysters. Collected 2 x 50mL from each treatment. Collected ~750mL from each treatment. Cells were pelleted 4000RPM, 15mins, 4C. Supe was removed and pellets frozen @ -80C.
100uL of a 1:1,000,000 dilution of the control bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. 100uL of a 1:10,000,000 dilution of the exposed bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. Control colony count the next day = 563 colony forming units (CFU). Exposed colony count the next day = 367 CFU.
Control Calculations
563 CFU/100uL = 5.63 CFU/uL
5.63 CFU/uL x 1:1,000,000 dilution = 5.63 x 10^6 CFU/uL
5.63 x 10^6 CFU/uL x 1000uL/mL = 5.63 x 10^9 CFU/mL
Exposed Calculations
367 CFU/100uL = 3.67 CFU/uL
3.67 CFU/uL x 1:10,000,000 dilution = 3.67 x 10^7 CFU/uL
3.67 x 10^7 CFU/uL x 1000uL/mL = 3.67 x 10^10 CFU/mL
500mL culture was split evenly between two containers containing 3L sterile sea water each. One container also contained 3 large, autoclaved C. gigas. Containers had an air stone to promote circulation. 4 x 1mL samples were collected from each container, pelleted @ 10,00RPM 1min. Supe removed and samples stored @ -80C. Samples will be collected @ t = 0, 0.5, 1.0 and 24 hrs. Containers were covered with aluminum foil to minimize splashing caused the by the air stone.
100uL of a 1:1,000,000 were plated on 1x LB + 1% NaCl plates and incubated O/N @ 37C. Colony count the next day = 399 colony forming units (CFU).
399 CFU/100uL = 3.99 CFU/uL
3.99 CFU/uL X 1:1,000,000 dilution = 3.99 x 10^6 CFU/uL
3.99 x 10^6 CFU/uL x 1000uL/mL = 3.99 x 10^9 CFU/mL
3.99 x 10^9 CFU/mL x 250 mL/container = 9.975 x 10^11 CFU/container
One of the two starter cultures from yesterday were used to inoculate 500mL 1x LB+1% NaCl and incubated O/N @ 30C 200RPM.
2X 10mL starter cultures of in 1x LB+1% NaCl were inoculated with two separate colonies of V. tubiashii and incubated O/N @ 30C 200RPM.
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.
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.
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.
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).
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.
