Have you tried Trizol method

Hello Linda Ratjama

Efficient extraction of most proteins in the tumor specimen is an important step. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors.

You may prepare RIPA buffer (1X) as follows.

50mM Tris-HCL, pH 7.6

150mM NaCl

1% NP-40 or Triton X-100

0.5% Sodium deoxycholate

0.1% SDS

1mM EDTA

Add protease inhibitor cocktail fresh (1X) or use the following protease inhibitors such as Pepstatin A (1uM), Leupeptin (10uM) and PMSF (0.1mM) when you are about to perform tissue lysis. Phosphatase inhibitors will be required to be added in the lysis buffer fresh only when phosphorylation states (activation states) are being investigated. The phosphatase inhibitors include Sodium fluoride (1mM) and Sodium orthovanadate (1mM). Perform tissue lysis below 4 degree C using cold RIPA buffer.

While for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred, cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA.

You may also consider using Urea buffer as it is another versatile and efficient cell and tissue lysing buffer.

Typical composition of Urea buffer include:

TRIS base 40 mM, pH 7.6

Urea 5M

Thiourea 2M

NP-40 or CHAPS 4%

DTT 10 mM

The additive thiourea present in Urea buffer can dramatically enhance the solubility of a wide range of proteins such as nuclear, membrane, cytosolic, including tubulin. Urea inactivates proteases that degrade cellular proteins. So, there is little need to add protease inhibitors during lysis.

Protocol in brief:

Good Luck!

If you have the money, it's worth buying the Qiagen kit that does DNA, RNA, and protein. Otherwise, you'll have to carefully split your samples and use 2 different methods (kit for DNA & RNA), another protocol/kit for the protein.

Honestly, the samples are so precious, I'd insist on the expensive kit.

Pls refer protein hand book protocold

Identify your protein source and localization--animal, plant or bacteria

For extraction--note down the weight of the sourec, use homogenize buffer, --Tris Hcl etc, Centrifuge at10k &t 4 degee C for 15- 20 minutes, collect the supernatant and do protein assay--Lowrys or Brad ford s method

For purificatio-- DIalysis, amm sulphate saturation, chromatography, autoanalyzers, electrophoresis and western Blot can be performed

You could do chromatography depending on the nature of the protein. However, you could get all the proteins in the by lysing it with RIPA buffer as mentioned above.

For protein extraction from laryngeal cancer samples without using a specialized protein extraction kit, you can utilize a common method called the TRIzol method, followed by protein precipitation using organic solvents. Here's a step-by-step protocol for your situation:

Materials:

Procedure: