4 replies to this topic

[Bill Nye]

I had a problem with low protein concentration in westerns so after a bit of tweaking I came up with a working protocol that helped. But now, maybe my protein concentration is too high (all my bands show up very dark)... lol so I'm just wondering if you guys have any advice for me.

for 60mm plates, I use 200ul lysis buffer

- aspirate media
- 1x wash with cold pbs
- add lysis buffer and scrape into cold microcentrifuge tube
- homogenize with needle (21 gauge, 20 pulls) on ice
- incubate at 4degrees with shaking for 20mins
- boil 5mins @ 100degrees
- spin 10mins @14k RPM
- load into gel / store at -20degrees

Do you see anything wrong with the steps? Would love some input, thanks!

[bob1]

try counting the cells before lysis and resuspend at 10⁴ cells per ul

[laurequillo]

Normally for direct lysis in laemli buffer I add the laemli buffer (120ul per sample 6well/plate), boil two minutes, then sonicates the samples (2x 15sec) and boil again 5 minutes. After that I directly load the samples.

If you dont use laemli buffer directly: I use lysis buffer for 15 min, the I centrifuge the samples for 15 min 13.000rpm, and then I add the laemli, boil the samples and load the gel

Edited by laurequillo, 25 September 2009 - 04:09 AM.

[icome]

You could try diluting your samples serially, such as 1:10,  1:8, 1:4, and 1:2, and then finding which dilution ratio is optimal.
I always lyze cells with lysis buffer (such as NP-40 in HEPES) and measure the protein concentration by bradford assay. Then I mix samples with 4X laemmli buffer and load them at 30-50 ug per well in SDS-PAGE. This may work better.

[Julie123]

icome, on Oct 8 2009, 03:53 AM, said:

You could try diluting your samples serially, such as 1:10,  1:8, 1:4, and 1:2, and then finding which dilution ratio is optimal.
I always lyze cells with lysis buffer (such as NP-40 in HEPES) and measure the protein concentration by bradford assay. Then I mix samples with 4X laemmli buffer and load them at 30-50 ug per well in SDS-PAGE. This may work better.

Can I add laemmli buffer directly to a 6 well plate?I dont need to scrape?would 120ul of lammeli be enough for a 1 well of 6 well plate if cells were confluent(90%)?