Common Solutions.

• RIPA Protein lysis buffer

• TAE Buffer Stock Solution, 50 x.

• PBS (1x)

• 6X SDS-PAGE Sample Buffer (with DTT)

RIPA Protein lysis buffer

final concentrations

150mM NaCl

50mM Tris pH7.3

0,1% SDS

1% NP-40

0,5% Natrium-Deoxycholat

1mM Natrium ortho vanadat pH 10 (pre-activated)

1mM EDTA

For 100ml:

5M NaCl                                                 3ml

1M Tris pH7.3                                         5ml

10% SDS                                               1ml

NP-40                                                    1ml

5% Na-Deoxycholat                                10ml

100mM Natrium-ortho-vanadate               1ml

500mM EDTA                                         0,2ml

dH2O                                                     78,8ml

·         store at 4°C

·         Na₄VO₃ might also be added just prior to use

·         Add PMSF prior to use
(1 tablet Roche Complete Mini Protease Inhibitor for 10ml or 40µl 25x PMSF for 1ml )

Pre-Activation of Na₃VO₄ for maximal inhibition of phosphotyrosyl phosphatases

·         Prepare 100mM Stock of Na₃VO₄

·         Adjust pH to 10.0 with NaOH or HCl. At pH 10 the solution will be yellow.

·         Boil the solution until it turns colorless

·         Cool to RT

·         Readjust the pH to 10.0  and repeat boiling and cooling until the solution remains colorless and the pH stabilizes at 10.0

·         Store aliquots at -20°C

·         This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of phosphatases

Procedure:

·         All steps should be performed on ice!

·         Homogenize Sample in RIPA (+Na₃VO₄ +PMSF) or add RIPA to PBS-washed cell layer, incubate 5min and scrape off cells, transfer to 1,5ml tube

·         Incubate on ice for 30min for efficient lysis

·         Centrifuge 10.000xg 4°C for 10min

·         Transfer supernatant to new tube and store at -20°C

TAE Buffer Stock Solution, 50 x

add dH₂O to 500 ml total volume

PBS (1x)

Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X) liquid
Contains no calcium or magnesium.

6X SDS-PAGE Sample Buffer (with DTT)

other variants:

Sample buffer 2 x, 10 ml:
Stacking gel buffer 4 x 2.5 ml
SDS, 10 % (cat.no. 20763 as 10 % solution in water) 4.0 ml
Glycerol (cat.no. 23176) 2.3 ml
2-Mercaptoethanol (cat.no. 28625) 0.5 ml
Bromophenolblue (cat.no. 15375) 1.0 mg

SDS-PAGE sample buffer, 4X, pH 6.8

0.25 M Tris

8% SDS

40% Glycerol

0.04% Bromphenol