Jacobs:Protocol Alkaline Phosphatase Assay: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
An M. Nguyen (talk | contribs) |
An M. Nguyen (talk | contribs) |
||
| Line 21: | Line 21: | ||
#* 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample | #* 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample | ||
#* Prepare enough of the working buffer for the standards and unknowns | #* Prepare enough of the working buffer for the standards and unknowns | ||
# Standards | # Standards | ||
#* Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase. | #* Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase. | ||
# Samples | # Samples | ||
#* Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul. | #* Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul. | ||
Revision as of 13:30, 17 February 2011
Alkaline Phosphatase Assay
Materials
- -20 freezer
- PMSF solution
- Protease inhibitor cocktail
- Control enzyme
- Substrate
- Fridge
- Diluted Triton buffer
- Miscellaneous
- 96 well plate
- 1.5 ml microcentrifuge tubes
- Pipette, tips
Preparation
- Buffer
- 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample
- Prepare enough of the working buffer for the standards and unknowns
- Standards
- Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase.
- Samples
- Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul.
Assay
- Prepare the wells by adding 20 ul of each standard or sample in triplicate.
- Add 180 ul of the working buffer to each well.
- Add 1 ul of the substrate to each well. Do this quickly as the substrate will immediately be converted.
- Measure fluorescence in the plate reader at 330 nm excitation and 440 nm. Generate a standard curve of FSU vs. alkaline phosphatase in ug/ml.
Notes
- 11/2/10 - Triton-X works much better than RIPA. RIPA may affect the protein-protein interaction.
- 11/15/10 - Colorimetric assay version of this protocol using substrate buffer and p-nitrophenol did not work well with MC3T3s, MLOY4s or primary osteoblasts. It is likely because it is not as sensitive as the fluorimetric assay.
