Preparation of Cell extracts for Western Blotting? - (May/24/2005 )
Hi, I am planning to carry out a Westen Blotting experiment to observe the translocation of NF-KB in CAC0-2 cells once I have stimulated them with various bacteria (including probiotics). Does anyone know a good protocol to prepare the nuclear cell extacts for this without the use of a kit? I have found a few methods but Im not sure if they would be suitable, any help would be much appreciated! thank you.
this one works in my hands
But sometimes, i get par of pellet in the eppendorf. So i decided always centrifuge max speed (at least 20 000g) for 5' at RT before the quantitation.
Wash cells with cold PBS twice
Add 4ml PB and scrap them (or trypsinization)
Pellet by 5’ 1000rpm 4°
Transfert in eppendorf
Pellet 2000 rpm 4° 5’
Resuspend pellet in twice the pellet volume of cold A buffer supplemented by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M
Add 0,05%NP40 after added Abuffer in all samples. Pellet 15’ 2500g 4°
Supernatant contains cytoplasmic proteins
Resuspend pellet in 1volume of B buffer supplemented by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M, anda dd 2/3 of volume of C buffer supplemented too by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M
Roll them at 4° for 45’
Pellet at max speed (at least 20000g) for 45’ at 4°
Supernatant contains nuclear proteins
Store at -80°
Proteins are ready do use.
Hypotonic A Bufer Pour 50ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 10mM 167µl
Tris pH 7.9 (1M) 10mM 500µl
Hypotonic B Bufer Pour 50ml
Glycérol 20% 10ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 10mM 167µl
Tris pH 7.9 (1M) 20mM 1000µl
Hypertonic C Bufer Pour 50ml
Glycérol 20% 10ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 1,2M 20ml
Tris pH 7.9 (1M) 20mM 1000µl
Thank you for your help.
Sorry, I am a bit unsure about what PIC is? can you please tell me and if there are any alternatives I may use to this if I can not find it avaliable. Thank you.
hi
i think PIC stands for protease inhibitor cocktail, which you can get it from sigma or roche. if the PIC is from sigma (choose the one specific for tissue culture). i ususally add 10ul to 1ml of my lysis buffer