General protocol for WB (RIPA Buffer)

RIPA buffer

• 50 mM Tris pH 8.0
• 150 mM NaCl
• 1% NP-40
• 0.5% sodium deoxycholate
• 0.1% SDS

Note
PI … proteinase inhibitor
PPI … phosphatase inhibitor

Reagent and equipment before you start

• RIPA buffer + additives (PI, PPI; if nessesary)
• Cool centrifuge
• Cool Bioruptor + vials for bioruptor (they are suitable for centrifugation)
  
• Cells can be directly harvest with RIPA buffer
• Trypsin for harvest
• medium
• Cool PBS
• Blocking
  • 2.5 - 5 % milk/BSA-T/TBS-T … depends on proteins; usualy TBS-T for phosporylated proteins, it is the most gentle

Protocol

Sample preparation

• WORK ON ICE
• mix RIPA buffer (+ PI + PPI)
• harvest cells in 1x PBS and transfer to a 1.5 ml Eppendorf tube
• Optional but recommended: count cells; depends on harvest
• centrifuge (3.000 rpm; 5 min; 4 °C)
• 2 x wash pellet with cold PBS; *this step depends on harvest
• Stop point 1: pellet can be stored at -80 °C; than slowly thaw on ice
• resuspend pellet in RIPA buffer + PI + PPI (approx. 80 μl/106 cells; 50 μl - 200 μl)
• incubate on ice for 30 min
• Note: SDS should be dissolve – check, if not, keep at r.t. for < 5 min
• sonicate (1 x 15 s)
• centrifuge (10.000 rpm; 10 min; 4° C)
• collect supernatant in new tube as “whole cell extract”
• Stop point 2: store samples at -20°C

Quantification of protein

• Quantification of protein
• Excel sheet for calculation (loading 30 – 50 μg of proteins; or depends on experience)

PPA gel

prepare in advance

Get important information about antibodies

Loading controls - options:

Protein	Host animal	Size [kDa]	dilution	SDS-page	cat.#	lot#
TFII H		100
Lamin B1		55
Vimentin		46
Actin		45
Tubulin		40
GAPDH		37

Trick : let mix roll in the falcon (3 min; r.t.) before adding APS/TEMED; gels will run nicer and more equally Ladder: 5 – 10 μL; 5 μL for 12% gel, 10 μL for 6% gel; stored in -20°C, thaw before use

ELFO and transfer

• add 6x lämmli buffer based on Bradford (Stop point 3: store samples mixed with lämmli at -20°C)
• boil (95°C; 5 min)
• cooled down to r.t.;
• spin down (500 g; 1 min; r.t.) just to collect all liquid condensates
• ELFO (approx. 1 – 2 h; 80V stacking gel, 140V resolving gel)
• transfer
  • wet transfer Biorad:
  • semidry transfer: depends ….
• Optional: 3 x wash 10 min
• Blocking 1 h; 5% BSA/TBS-T
• Primary antibody in 5% TBS-T(O/N; 4°C)
• 3 x wash 10 min
• Secondary antibody in 5% TBS-T(2h; r.t.; fluorescent)
• 3 x wash 10 min

Secondary antibodies

• HRP 1 : 10 000
• Fluorescent 1 : 15 000

Detection