Enzyme Explorer

CPS Flag


Detection of FLAG-tagged (FLAG-BAP) protein in an E. coli K12 extract.

Procedure:

The procedure and illustration below demonstrate detection from 10 nanograms down to 40 picograms of the FLAG-BAP control protein loaded on a 4-20 % Tris-Glycine SDS-PAGE gel utilizing the CPS-1 substrate.


1. The example below utilizes 4-20 % gradient SDS-PAGE gels with 1X Tris-glycine buffer (T 7777). The FLAG-BAP samples were diluted with Sample Buffer, Laemmli 2 Concentrate (S 3401). The Sample Buffer 2X For FLAG-Tagged Protein Immunoprecipitation (S 8684, nonreducing) works also.
2. The samples were heated on a 100 °C heating block for 4 minutes.
3. 20 µl of each serially diluted sample, including 2X sample buffer, were added to each well. The amount of FLAG-BAP in each well is shown below.
4. The gels were run at 145-155 Volts for 1.5 hours or until the dye front reached ~1 cm above the end of the gel.
5. Blotting pads and paper were pre-wet in Towbin’s buffer (100 ml T 4904, 200 ml methanol, 700 ml deionized water).
6. PVDF membranes were pre-wet in methanol for 3 minutes, then in water until ready to transfer the gel.
7. The SDS-PAGE gels were washed in deionized water for 2 minutes.
8. Pre-wetted pads, papers, and gels were placed into the Western blotting cartridge. The membrane was placed over each gel. Any air bubbles can be eliminated by rolling a glass pipette over the membrane.
9. Towbin’s buffer (and ice tray ) were placed into the apparatus.
10. Transfer was run at 65-75 Volts for 1 hour.
11. The blot was washed in deionized water (at least 0.5 ml/cm) for 1minute with mild agitation.
12. Blots were blocked with Western Blocker (W 0138) solution for 30 minutes. 15 ml of blocker should be used with each membrane.
13. A 1:300,000 dilution of rabbit anti-mouse IgG, peroxidase conjugate (A 9044) was used for the Sigma CPS-1 blot. Dilutions of 1:10,000 to 1:80,000 were required for the competitors’ substrate solutions. Membranes were incubated for 30 minutes.
14. TBST Buffer (Tris-buffered saline with TWEEN® 20 was made using contents of a TBST packet (T 9039) mixed with 1 liter of high purity water (W 4502)
15. The Sigma CPS-1 blot was washed with 20 ml of a TBST 5 times for 5 minutes each time. The competitors’ blots were washed with their own brand of wash solutions where required.
16. Membranes were drained of TBST and placed on a plastic sheet. 5 ml of substrate solution was added to each membrane. Excess substrate was removed after 5 minutes; however, the membranes were kept moist.
17. Membranes were placed in plastic sheets and exposed to film for 30 seconds. The film was placed in developer for 45 seconds, then washed and placed in fixer for 45 seconds. The film was rinsed and then scanned.

The FLAG-BAP control protein was diluted in cell extract to a very low level (down to 0.008 nanograms per microliter) in order to demonstrate detection at low expression levels.
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