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In Topic: Sediments/debris/pellet in protein lysate?
15 March 2013 - 04:59 AM
almost a doctor, on 15 March 2013 - 01:49 AM, said:
What's your lysis buffer?
Could it be that your protein concentration is high and some of it has come out of solution and precipitated? This are usually hard to get back into solution.
Just a thought
Could it be that your protein concentration is high and some of it has come out of solution and precipitated? This are usually hard to get back into solution.
Just a thought
I'm using a lysis buffer for phospho-proteins with the following:
Tris
EDTA
NaCl
Triton-X
NaF
Na deoxycholate
Na2VO3
Protease inhibitor cocktail
This is the first time seeing observable amounts of sediments in my samples. Could it be that I've stored my sample for too long at -20C?
In Topic: Western blot- bands weak
01 March 2013 - 04:24 PM
sdheeru, on 01 March 2013 - 04:04 PM, said:
Earlier I used to put ECL for 1 min and then keep the membrane in the cassette. Then I tried for 30 s as he does, I don't see any improvement (either bands are weak or bg is high).
What do you mean you keep the membrane in the cassette? How did you probe your membranes with the primary & secondary antibodies - I do mine in small clip-lock plastic food containers. Same question would also go with how did you wash your membranes - in the cassette?
sdheeru, on 01 March 2013 - 04:04 PM, said:
-The cassette for developing the blot. He has white cassette (from inside) and mine is black.
-PVDF wetting time.
-The side of gel used against the membrane and ultimately upside for the antibody solutions.
Do you think these factors could be a cause??
-PVDF wetting time.
-The side of gel used against the membrane and ultimately upside for the antibody solutions.
Do you think these factors could be a cause??
Does 'developing' mean transfer?
A rule of thumb, the sequence of your transfer 'sandwich' cassette is:
black side (-), sponge/fiber pad 1, blotting paper 1, gel, membrane, filter paper 2, sponge/fiber pad 2, white side (+). So when you open your cassette after a transfer, your membrane will be on the white cassette.
Check the nice picture to represent what I described here: http://www.abcam.com...ource&rid=13045
QUESTION:
1. Did you run any protein ladder/markers?
2. What was your protein of interest?
3. Did you probe the same membrane for housekeeping proteins/loading controls - e.g. actin, tubulin, GAPDH, histone H3?
