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There have been 14 items by kegaff (Search limited from 30-May 19)
So just wondering, am i messing up any step here? Or what could be a best solubilizing agent for low mw protein such that I can do Bradford after tca ppt also to know how much protein is going in?
After extraction, when i did tca precipitation, i washed with acetone and directly added tris trcine sample buffer from biorad. The protein sample was still blue and not yellow so i didn't do any neutralization. However, it was too difficult to re suspend the protein in sample buffer so i incubated at 37 for half an hour. The easily dissolved low mw protein was directly loaded even though there was undissolved protein in the tube.
So currently i am using 10-20% tris tricine buffer from biorad and sample buffer and tris tricine running buffer also from biorad with protein marker that can show even 1 kb band.
I did a semi dry transfer at 10v for 40 mins. Its a gradient gel, so i was not too much worried about that longer transfer. I could see 2kb ladder band too in my membrane. I haven't stained the membrane though. However, i am staining the gel and it looks pretty clear with less residue in there.
I am doing imaging in few hours 3-4 hours. Let's see how it turns out. But i have few questions regarding my protein sample.
I had 2 sets of protein sample. For half i did, no treatment for the other half i did TCA precipitation. When I was running the gel, the sample front moved down for tca precipitated ones while the other without precipitation did not actually moved away as the front. It made a kind of floral patch at 10 kb area. So i am thinking that for non treated sample its going to mask with the band, if there are any
So, my question is do we have to do tca precipitation for small protein sample. My size is 6.9kb with flag tag.
Can we do Bradford assay after tca precipitation because i am hoping that my quantification of protein before tca precipitation can go off after my precipitation?
I have even read somewhere that people autoclaving the membrane after transfer. I am hoping that they do it to fix the protein in the membrane. Is it done specifically for ubiquitin or can be done for any small protein?
I have a problem detecting the small protein of 7kb in western blot. Hers is the procedure that i have been using
my protei has two isoforms of 37KD and 7KD with the flag tag. some other people have already show i different system and we believe it occurs in two isoforms in our system too looking at the protein sequence.
i have been using biiorad precast tris glycine gel gradient of 4-20%. run at 180V for 45 mins. do semidry trasfer at 10V for 1 hr in 0.2micronm PVDF. i use PBS buffer and wash in PBST. the marker in the membrane are quite visible but when i do imaging, the 37kD protein only gets detected. somewhere in the forum, i saw heating the membrane in boiling water to fix the small mw protein, i tried that too but i could see nothing at 7kb band are.
What is the suggestion for doing western blot for small MW protein like 7kb?