Sample Preparation

For sample preparation mechanical and chemical techniques can be employed. Tissue homogenizers (potter, tissue mills, blenders) and lysis buffers like RIPA, TritonX-100/NP40 buffers break up tissue or cell structure and solubilize proteins.

Mild extraction buffers like detergent free HEPES/Sucrose, TritonX-100 or NP40 buffers preserve protein-protein interactions but show poor extraction of tightly bound membrane or cytoskeletal proteins. Stronger buffers (e.g., RIPA, SDS‑containing formulations) release those proteins at the cost of disrupting some complexes.

Therefore, it is important to choose the appropriate lysis buffer for your protein of interest.

Protease and phosphatase inhibitor cocktails should be included to prevent protein degradation and to preserve the phosphorylation state.
In the following table you can find some of the most frequently used lysis buffers for sample preparation.

Lysis buffer

Buffer	Composition	Comment
RIPA	50 mM Tris-HCl, 150 mM NaCl, 1% TritonX 100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4	Strong solubilization of membrane‑bound and nuclear proteins; retains many phospho‑epitopes; compatible with downstream SDS‑PAGE.
NP‑40 lysis buffer	50 mM Tris‑HCl, 150 mM NaCl, 1 % NP‑40, pH 7.4	Milder than RIPA – preserves protein‑protein interactions; good for co‑IP of soluble complexes.
Triton X‑100 lysis buffer	50 mM Tris-HCl, 150 mM NaCl, 0.5 % Triton X‑100, 1 mM EDTA, pH 7.4	Low detergent concentration → minimal denaturation, good for membrane‑protein enrichment.
CHAPS buffer	40 mM HEPES, 150 mM NaCl, 1 % CHAPS, pH 7.5	Zwitterionic detergent – solubilizes membrane proteins while preserving native complexes; compatible with Native PAGE.
HEPES-Sucrose buffer	4 mM HEPES, 320 mM Sucrose, pH 7.3	Only mechanical homogenization, protein interactions stay intact. Suitable for soft tissues like brain.

As a final step ad 2x to 4x Laemmli SDS sample buffer to a 1x final concentration. To break up disulfide bonds, add a reducing reagent like mercaptoethanol (2 % final concentration) or DTT (50 mM final concentration). For complete denaturation heat the sample for 10 min at 70°C or for 5 min at 95°C.

Exception: many trans‑membrane or highly hydrophobic proteins aggregate upon heating. For these proteins, skip the heat step and load the samples unboiled (the SDS and reducing agent are usually sufficient for denaturation).

Ideally 10-30 µg of total protein (cell or tissue lysates) should be loaded per lane.