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.