SDS PAGE optimization

Multiple attempts at pouring 6-10% stacking gels or 10% SDS-PAGE gels were attempted. Consistently, commercial 10% NuPAGE (Invitrogen) gels provided better sample separation and clarity. Figure 2.2 shows a representative experiment where human seminal plasma (30µg) and mock- transfected 293T media (10µg) were separated in a 10% NuPAGE and a 6-10% poured stacking gel in the same electrophoresis chamber. Post electrophoresis, gels were stained using the same coomassie stain and destain. Therefore, the only difference between gels was the preparation (poured or commercial).

Figure 2.2: Commercial NuPAGE gels yield better clarity than poured stacking gels Mock- transfected 293T media (10µg) and human seminal plasma (30µg) were separated in a 10% NuPAGE gel (left) and a 6-10% poured stacking gel (right) and stained with coomassie. The commercial NuPAGE gel (left) yielded better separation of samples.

2.3.3 Western blot optimization

2.3.3.1 Bio-Rad wet transfer system provides band clarity in Western blots compared to iBlot™ transfer system

Polyclonal rabbit anti-SEMG1 and anti-SEMG2 antibodies were ordered from ABCAM, and SEMG2 primary antibody was used to optimize several detection methods. An iblot™ (Invitrogen) dry transfer system was temporarily available in our department for trial

experiments. The iblot™ system transfers protein from an SDS-PAGE to a membrane within 7 minutes, whereas our Bio-Rad wet system transferred proteins between 45-75 minutes. Human and chimpanzee seminal plasma (20µg) in quadruplicate was separated on the same SDS-PAGE gel. Then the gel was cut into equal halves and proteins were transferred utilizing either the iblot™ system or the Bio-Rad wet transfer system. Post-transfer the PVDF membranes were incubated in antibodies and washes in the same container. The only difference between

NuPAGE Poured gel

293T Hu#4 Mock 293T Hu#4 Mock kDa 198 98 62 49 38 28 17 kDa 98 62 49 38 28

membrane detection was the transfer system. When using the anti-SEMG2 (ABCAM) primary antibody, Bio-Rad wet transfer yielded cleaner results with FastRed detection (Figure 2.3). Distinct bands around 70kDa were detected for human seminal plasma using the wet transfer system, while the iblot™ transfer system did not detect the full size of SEMG2. It is important to note that SEMG proteins are known substrates for prostate-specific transglutaminase and for Kallikrein 3, and the smearing pattern of protein is probably a result of protein cross-linking and degradation.

Figure 2.3: Bio-Rad wet transfer system has a clearer signal to background Western blot ratio compared to iBlot™ dry transfer

Human and chimpanzee seminal plasma (20µg) was separated on the same SDS-PAGE gel in quadruplicate, transferred by the iBlot™ dry transfer system or the Bio-Rad wet transfer system, and incubated with the same antibodies and FastRed detection substrates.

2.3.3.2 Non-specific bands are due to cross-linking and degradation of seminal proteins and not due to our Western blot protocol

In order to address the smeary appearance of our seminal plasma Western blots (seen in Figure 2.3), we used our protocols and equipment to blot samples that are not known to be cross- linked or degraded. Hospital samples of nonglycosylated Pseudomonas aeruginosa isolates (B017, B018, and B029) were gifted from Dr. Pete Castric’s lab, along with their optimized

iBlot Wet Transfer

Hu #4 Ch#2 Ch#2 Hu#4 Hu #4 Ch#2 Ch#2 Hu#4 kDa 198 98 62 49 38 28 17 kDa 198 98 62 49 38 28 17

monoclonal mouse anti-pilin primary antibody. Our lab equipment and FastRed Western blot protocol were utilized with their samples and primary antibody. Nonglycosylated mature pilin protein is around 16 kDa molecular weight while glycosylated pilin protein is around 17kDa (Castric et al., 2001). Nonglycosylated pilin samples were detected at the expected 16kDa with low background signal (Figure 2.4). This Western blot confirmed that our equipment and

protocol were sufficient for detection of protein using optimized working antibodies. In addition, if the protein of interest (pilin) was not susceptible to degradation and cross-linking (unlike SEMG1 and SEMG2), our methods would detect clean single bands within the expected molecular weight range.

Figure 2.4: Nonglycosylated pilin was cleanly detected from hospital samples

Pseudomonas aeruginosa isolates (B017, B018, and B029) from Children’s Hospital of Pittsburgh were separated

on an SDS-PAGE and mature nonglycosylated pilin was detected around 16kDa. kDa 198 98 62 49 38 28 17 14 B029 B018 B017 Anti-Pilin

2.3.3.3 Alkaline phosphatase conjugated secondary antibody and FastRed colorimetric detection reduces signal to noise ratio compared to chemiluminescent detection

I wanted to assay the sensitivity of chemiluminescent detection with horseradish peroxidase (HRP) conjugated secondary antibody and the FastRed colorimetric detection with alkaline phosphatase (AP) conjugated secondary antibody. For this experiment, purified human actin (Sigma Aldrich) with different amounts of protein (15ng, 31ng, 62.5ng, 125ng, 250ng, 500ng, and 5,000ng) were separated and transferred to PVDF membranes under the same conditions. The only difference between membranes was the secondary antibody applied (either HRP or AP conjugated) and the detection substrate (ECL chemiluminescent or FastRed

colorimetric, respectively). Both detection methods were able to detect 250ng of actin (Figure 2.5), but the FastRed colorimetric Western blot had relatively less background noise compared to signal.

Figure 2.5: Colorimetric FastRed detection has reduced background noise compared to chemiluminescence

Human actin protein was Western blotted using horseradish peroxidase (HRP) conjugated secondary antibody and chemiluminescent ECL substrate and imaged on the typhoon (left) and was also Western blotted using alkaline phosphatase (AP) conjugated secondary antibody and FastRed colorimetric detection (right). Lanes are labeled with total amount of protein that was loaded into the SDS-PAGE gel. Both detection methods were able to visualize actin in greater amounts than 125ng.

15ng 31ng 62.5ng 125ng 250ng 500ng 5µg

15ng 31ng 62.5ng 125ng 250ng 500ng 5µg

Chemiluminescence FastRed Colorimetric