Protein Biochemistry
Native gels
AG Dohmen 2013
University of Cologne
Reference: Elsasser, S et al. (2005) Characterization of the proteasome using native
gel electrophoresis 398, 353-363.
Stock solutions:
1. 450 mM Tris + 450 mM boric acid (5 fold stock solution), do not need to adjust
pH. pH should be 8.3
2. 1 M MgCl2 (200 fold)
3. 0.5 M EDTA, pH 8.0 (1000 fold)
4. 250 mM ATP + 250 mM MgCl2 (250 fold) + 500 mM Tris base. Freeze in
aliquots and store at -80oC. Thaw only once.
5. Sample buffer 5x (250mM Tris-HCl (pH7.5) 50% glycerol, 60 ng/ml xylene
cyanol). Freeze at -20°C.
Gel preparation (should polimerize in 10 min)
3.5% acrylamide, 90mM Tris, 90mM boric acid, 5mM MgCl2, 0.5mM EDTA,
1mmATP-MgCl2, 0.1%TEMED,0.1%APS
total volume
acrylamide stock (Roth)
Tris-boric buffer (5x)
MgCl2 (200x)
EDTA (1000x)
ATP- MgCl2 sol. (250x)
H2O
10%APS (100x)
TEMED
15 ml
1.75 ml
3.0 ml
75 ul
15 ul
60 ul
1.5 ml
150 ul
15 ul
40 ml
4.7 ml
8.0 ml
0.2 ml
40 ul
160 ul
26.46 ml
400 ul
40 ul
Running buffer
500 ml
Tris-boric buffer (5x)
100 ml
MgCl2 (200x)
2.5 ml
EDTA (1000x)
0.5 ml
ATP- MgCl2 solution (250x)
2.0 ml
H2O
to 500 ml
1000 ml
200 ml
5.0 ml
1.0 ml
4.0 ml
to 1000 ml
Protein Biochemistry
Running conditions:
1. Wash away all residual SDS from your plates, supports, clamps, combs and
running chambers with plenty distilled water.
2. Prepare mini-gels at room temperature. Use 1.5 mm spacers. After polimerization
let them cold down at 4°C.
3. The samples should be kept on ice all the time.
4. Run the gels at 4°C or on ice at 100V (23-25mM) for 3 hours.
5. After separation stain proteins with silver, detect proteasome with overlay assay
or incubate the gel 20min in Transfer buffer containing 2% SDS and transfer the
proteins 2h at 0.8mA/cm2 .
Protein concentrations for fluorogenic assay
1 – Purified proteasomes: 1-5 ug/mm lane width, we will use combs with 10 lanes
(around 5mm), so apply at least 5ug of purified proteasome in a well
2 – total protein from a crude extract: 50-300ug of total protein per lane.
3 – To detect proteasomes by immunoblot 10ug-20ug of total protein from a crude
extract should be enough, depending on the antibody one uses.
Overlay activity assay (Elsasser et al, 2005)
1 – After electrophoresis incubate the gel in 50mM Tris(pH7.4), 5mM MgCl2, 1mM
ATP, for 5 minutes.
2 – Decant the solution.
3 – Add 50uM suc-LLVY-AMC in 50mM Tris(pH7.4), 5mM MgCl2, 1mM ATP for 30
minutes and incubate at 30°C. Be sure taht the gel is flat and completely covered.
4 – Expose the gel to UV light and photograph it.
or (it works better in Roshini’s hands)
In-gel degradation assay: (R. Verma et al., MBC,2000)
Incubate the gel in 0.1 mM fluorogenic peptide substrate Suc-LLVY-AMC for 10 min
at 30◦C in a petridish
Solution: 10% glycerol, 25 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM ATP, 1 mM
DTT
Stock
Working
Volume of stock
75% glycerol
10% glycerol
1.3 ml
1M Tris-HCl
25 mM Tris-HCl
250 µl
1M MgCl2
10 mM MgCl2
100 µl
100 mM ATP
1 mM ATP
100 µl
1M DTT
1 mM DTT
10 µl
Suc-LLVY-AMC
100 µM (76.3 µg/ml)
76.3 µl
10 mg/ml
H2O
8.2
Total
10 ml