Flow Cytometry: Flow cytometry analysis using anti-phosphoserine, clone 4A4. Cells were either untreated (shaded, green) or treated (unshaded) with Calyculin A/Okadaic acid (30 minutes). Analysis was run with mouse IgG1 control (purple).

Immunofluorecence Microscopy:
Confocal immunofluorescence image of insulin-treated 293 cells labeled with Anti-phosphoserine, clone 4A4 (green) and DAPI (blue).

Phosphoserine Phosphospecificity and Affinity using Luminex®:
Luminex® assay using either phosphorylated serine or non-modified serine to determine anti-phosphoserine, clone 4A4 affinity and phosphospecificity.

Detection of Ser/Thr Kinase by Western Blot:
Anti-phosphoserine, clone 4A4 was used to detect these 10 active kinases in denatured western blot conditions. Recombinant active kinases were resolved on SDS-PAGE and transferred to PVDF membrane. The membrane was probed with anti-phosphoserine, clone 4A4 (2µg/mL) in 3% milk. Note: some of the kinases were not full-length proteins, hence they run at a reduced molecular weight.

Detection of Inactive and Active Recombinant Kinases by Western Blot:
Anti-phosphoserine, clone 4A4 was used to detect the inactive and active counterparts of 6 recombinant kinases expressed by baculovirus in Sf21 cells in denatured western blot conditions. Recombinant active kinases were resolved on SDS-PAGE and transferred to PVDF membrane. The membrane was probed with anti-phosphoserine, clone 4A4 (0.5 µg/mL) in 3% BSA. Note: expression of various kinases by baculovirus in Sf21 cells can result in basal phosphorylation levels of various serines on the recombinant protein. It is important to note the increase in phosphorylation in the active counterparts. Some of the kinases were not full-length proteins and other with tags, hence they run at a different molecular weight from what would be expected by an endogenous protein of the same target.

Anti-Phosphoserine, clone 4A4 blotting conditions:
Appropriate blotting conditions are very important with the use of clone 4A4. It is crucial that the secondary antibody ALWAYS be in 3% milk; never BSA. Use of secondary in BSA will result in dirtier blot. It is also highly recommended to use 2 µg/mL of 4A4 in milk/TBST for most western blotting applications. We recommend only using clone 4A4 at 0.5 µg/mL in BSA when the target is particularly hard to detect. This figure shows the insulin treated 293T cells run in parallel and probed with anti-phosphoserine, clone 4A4 at either 0.5 µg/mL in 3% BSA/TBST (lane 1) or 2 µg/mL in 3% milk/TBST (lane 2). Both lanes used 3% milk/TBST for the secondary incubation.

Phosphospecificity Demonstrated Through Phosphatase Treatment:
50 ng of recombinant Akt (expressed by baculovirus in Sf21 cells) was either untreated (Lanes 1 and 3) or pretreated (Lanes 2 and 4) with lambda phosphatase. The two samples in duplicate, were resolved by SDS-PAGE and transferred to PVDF membrane. The membranes were then subsequently probed with either Anti-Akt, PH Domain, clone SKB1 (Cat. No. 05-591, lanes 1 and 2) at 1 μg/mL or Anti-Phosphoserine, clone 4A4 (Cat. No. 05-1000, lanes 3 and 4) at 2 μg/mL.

Phosphoserine Specificity Determined by Blocking Amino Acids:
Calyculin A/Okadaic acid-treated A431 cell lysate resolved by SDS-PAGE, transferred to PVDF membrane, and subsequently probed with anti-phosphoserine, clone 4A4 in the presence of various amino acids.

Immunoblot Analysis:
CalyculinA/Okadaic Acid untreated or treated A431 lysate (lanes 1 & 2, respectively) or Insulin untreated or treated 293 cell lysate (lanes 3 & 4, respectively) were resolved by electrophoresis, transferred to PVDF, and probed with anti-phosphoserine,4A4 (0.5 µg/mL). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system.