Hi, I'm trying perform BN-PAGE with thylakoid samples of cyanobacteria. However, my protein can not run into the gel at all and stacked in the Spotting holes. I dissolved samples according the following steps:(1) wash samples: Sample (200 μg protein or according to the need) + 500 μl washing buffer;
(2) remove supernatant: 20000g, 30 min;
(3)resuspend the pellet:add 10 μl (or according to the need) resuspension buffer to final pr. conc. 20μg/μl, 10min on ice;
(4)add same volume of detergent (3% DM), mix gently
(5)40 min on ice to dissolve the membrane, 18000 g, 10 min, leave the supernatant
(6)take the supernatant to a new EP add 1/10 volume of sample buffer and gently mix.
Here's some other info on my protocol:
Washing buffer
330 mM sorbitol
50 mM BisTris pH 7.0
Resuspension buffer 100 μl
50BTH40G 50 μl
Pefabloc (10 mg/ml) 2.5 μl
MgCl2 (1 M) 1 μl
RQ1 (DNase) 4 μl
H2O 42.5 μl
Solubilization buffer (3 % DM) 100 μl
50×BTH40G 50 μl
Pefabloc (10 mg/ml) 2.5 μl
MgCl2 (1 M) 1 μl
RQ1 (DNase) 4 μl
10% DM 30 μl
H2O 12.5 μl
Hi there,
For whatever reason they might not be negatively charged enough to enter the gel... But looking at the picture I think the proteins just precipitate in the wells. What is the composition of your sample buffer?
DDM was always somewhat problematic with BN PAGE in my hands.
I'd try to lower it - 0.02% DDM was enough in my case.
Also, adding some(0.05-0.1%) SDS and loading less sample
has often helped.
No, I work with very different proteins.
Once you dissolved the membranes,
cmc concentration of DDM is often enough.
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