Hi Maryline,

I can tell you what I used to do for maximum results.

Start by homogenizing your sample with a blender (or any other means available to you) in 1 or 1.5 ml Tris–HCl buffer (200mM, pH 8.0), also containing EDTA (3mM) and sodium dodecyl sulphate (SDS)(1%, w/v). SDS will inactivate enzymes by disrupting the non-covalent bonds.

Subsequently you should put your homogenized sample on ice for 2 min and heat for 5min at 100◦C.  Repeat this procedure twice in order to inactivate any possible proteolytic enzymes. Centrifuge your homogenized samples for 1 min at 10,000×g and transfer the supernatant to a new 1.5ml eppendorf tube. You can also consider not using the centrifuge step, in order to make sure you have not lost any possible proteins in your sample.

At this point you can measure the protein concentration by any means available to you and use the samples for SDS-PAGE and Western blot.

Good luck,

Ali

Hi Maryline,

I can tell you what I used to do for maximum results.

Start by homogenizing your sample with a blender (or any other means available to you) in 1 or 1.5 ml Tris–HCl buffer (200mM, pH 8.0), also containing EDTA (3mM) and sodium dodecyl sulphate (SDS)(1%, w/v). SDS will inactivate enzymes by disrupting the non-covalent bonds.

Subsequently you should put your homogenized sample on ice for 2 min and heat for 5min at 100◦C.  Repeat this procedure twice in order to inactivate any possible proteolytic enzymes. Centrifuge your homogenized samples for 1 min at 10,000×g and transfer the supernatant to a new 1.5ml eppendorf tube. You can also consider not using the centrifuge step, in order to make sure you have not lost any possible proteins in your sample.

At this point you can measure the protein concentration by any means available to you and use the samples for SDS-PAGE and Western blot.

Good luck,

Ali

Hi Maryline,

In addition to the extraction methods mentioned above, you can add a step to concentrate your samples using Centricon or similar concentration methods.

Good luck. Norbert

For small amounts of kidney samples like yours in the past  - like your  - for the purposes of Western blotting - I have simply snap frozen homogenised & then put the homogenised mass directly in to ice cold RIPA buffer + Protease inhibitors

Alternatively, if your gene is low copy number and you wish to keep your protein concentration high, such that you can load up to 30ug of total lysate into a single well (therefore in 10ul to 20ul) direct suspension of your homogenised mass in NuPage loading buffer + 2ME or DTT can also be effective although you might want to sonicate the resulting load buffer lysate or pass a few times through a fine gauge needle as high viscosity (linked to genomic DNA) can render loading difficult

Regardless, all above suggestions will also work well

you can digest it with enzymes followed by protein analysis

I guess the aim is to get "as much protein as possible", but not to concentrate it. So the key is to lyse the  tissue TOTALLY, no debris left. My suggestion is Laemmli sample buffer plus protease inhibitor cocktail and DTT. (The only problem is bubles from SDS.)

Reference: http://www.ncbi.nlm.nih.gov/pubmed/25852189

the method that i use for extracting maximum possible proteins is adding methanol to the macerated tissue and precipitating all proteins, centrifuge them at minimum 10000 rpm for 20 min. obtained pellet is redissolved in desired buffer or simply in 50mM Ammonium Bicarbonate solution.

this will give you all proteins plus there is no issue of concentrating after extraction

I remember seeing some very interesting technology using pressure cycling for efficient cell disruption.  I am not sure of which company (it's been several years) but I found the link below as one example.  With the right buffers (as described above) the pressure approach could be useful.

http://www.pressurebiosciences.com/downloads/sell-sheets/PCT-HD.pdf

I can only think about RIPA buffer which might be the most potent lysis buffer which even extracts out nuclear protein. Extract using beads method. Use Nanodrop to measure the protein content instead of regular protein assay kit. Use 6X Lammeli buffer (recipe will be available by Google it).

RIPA (incl Protease Inh) buffer would be best choice in my experience, however if you can snap freeze the sample in liquid nitrogen followed by mortor -pistle protein extraction. Freezing the sample help to lyse better and finally yield more protein. try using 100 mg tissue with 200-400 micro litre of RIPA.

All the very best

I agree with those that suggested RIPA

Try RIPA plus sonication in a eppendorf on ice , before the centrifugation steps. Short pulses to avoid the heating of your samples. Best! conpare the yield doing tge same process with mouse tissue, meassure the protein at OD260 or by BCA, I guess your sample is more important

Lysating the sample directly in loading buffer. Another option that works well is doing a normal lysis, then precipitating the protein of the sample with TCA, then resuspending the pellet with the volume of loading buffer you will use to load on the gel (20-50ul)

JP