The protocol which I have used for different tissues - heart, liver and kidney -

Homogenized (~ 200 mg) in protein extraction buffer (Tris-HCl, NP-40, NaCl, EDTA, NaN3, and PMSF at pH 7.5) with freshly added protease inhibitors (DTT, leupeptin and aprotinin) using mortar- pestle or using sonication method and centrifuge lysate at 27,000g for 20 min. Collect the supernatent and determine protein concentration to

using protein assay e.g. Pierce Protein Reagent Assay BCA Kit.

We have found this method consistent and seems to work for majority of tissues.

I tend to use the same technique of sonicating the organ in lysis buffer (RIPA or Tris Lysis Buffer) with protease buffers directly. This generally works for murine lungs, hearts, kidney, livers and intestine. Protein yield will certainly differ between tissues and depend on tissue mass, but can than latter be normalised for by BCA.

Yes, volume of buffer is certainly an issue. This can become a bit of trial and error. A good rule of thumb are the volumes mentioned in the NE-PER Kit from Thermo-Fischer for starters. This mentions the volume of buffer according to sample weight. I find this works well in general.

Snap freeze your sample after collection, when preparing the samples add lysis buffer with protease inhibitor and apply sonication. It worked well for me when I was homogenizing lymph nodes. Keep an eye on the temperature, e.g. 3x10s of sonication (cone sonicator) and in between 30sec (or more) on ice to cool down.

Not an easy task since every tissue is different so you will need to normalize the total amount of protein for each tissue once extracted. This is usually a pain. BCA assay tend to increase reading with time (at RT) and samples containing lipids display aberrantly high absorbances and detergents as well as sulfhydryl reagents interact with the assay. Protein precipitation (deoxycholate-trichloroacetic acid) will remove most of interfering substances.

To prepare the samples the suggestions given by previous comments are all valuable but have you considered fractionation to partially purify your protein? Do you know if your protein is associated with membranes or is soluble? In such a case you might perform a subcellular fractionation and obtain a partial purification of your protein allowing an easier normalization of the amount of protein per sample. If it is soluble it is even easier. Anyway, if you will ever consider fractionation you can use the attached protocol [or only a part of it just to separate membrane proteins (P3) from soluble proteins (S3)] I used mainly for rat brain and torpedo electroplax (that are quite different) but also for a number of other tissues. If you want to perform the method remember not to use detergent before fractionation. :)

Hi Kai Albring,

Greetings.

I have been working for India's premier pharmaceutical Drug Discovery R&D biology since last 6 years as biologist. In my experience any tissues such as brain, liver, intestine, kidney, spleen etc simply you can use RIPA buffer without any doubt but which include protease inhibitors and DTT (if you work with Gel electrophoresis to).

Try to follow the below instructions I’m sure you will get

 Weight of tissue not more than 50 to 100mg

 Two ways you can use for extraction i.e using mortar- pestle method and mechanical method

 Mechanical method should not go beyond 20hz and 2 minutes

 Liver & heart gives more amount of protein so you can use more amount of RIPA buffer

Other tissues like Skeletal muscle it is very hard so

 Use Liquid nitrogen initially to make it power then follow the same protocol

I think it would solve your questions

You can reach me at anytime if you need any help

[email protected]

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+91-800-805-0303

I feel this paper might be of help as the similar comparison is performed here.

http://link.springer.com/article/10.1007%2Fs00018-010-0472-x

Thank you all for your suggestions! They will be very usefull for me to set uo the experiment.

And I have to say that I am really impressed how helpful it is to put questions like this on researchgate!

Best regards,

Kai

You can always try Trizol, it extracts RNA, DNA, and protein from all tissues, a dounce homogenizer is the best way to break open the cells in the presence of Trizol.

You can use the phenol extraction. You can get the highly purified proteins.

Here is the paper. http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048613

The protocol which I have used for different tissues - heart, liver and kidney -

Homogenized (~ 200 mg) in protein extraction buffer (Tris-HCl, NP-40, NaCl, EDTA, NaN3, and PMSF at pH 7.5) with freshly added protease inhibitors (DTT, leupeptin and aprotinin) using mortar- pestle or using sonication method and centrifuge lysate at 27,000g for 20 min. Collect the supernatent and determine protein concentration to

using protein assay e.g. Pierce Protein Reagent Assay BCA Kit.

We have found this method consistent and seems to work for majority of tissues.

A question for the previous post, can this be done in a non-chilled centrifuge?

RIPA buffer is more suitable for protein extraction from all kind of tissue..

I am impressed by the feeds because i have to extract RNA from the myocardium for my research. Greetly appreciate all the above feedz...

Protein lysis buffer (Tris-HCl, NP-40, NaCl, EDTA, NaN3, and PMSF at pH 7.5) with freshly added protease cocktail inhibitors then homogenization.  Centrifuge at 20,000g for 20 min. Collect the supernatent and determine protein concentration to

using protein assay, BioRad Kits

I've recently tried the ThermoFisher T-Per Tissue Protein Extraction Reagent and had great success for a number of murine tissues, regardless of whether the protein of interest was membrane-bound, cytoplasmic or nuclear. I would recommend comparing a few extraction buffers, but this has been the most consistent for me so far. Here is a link to the catalogue information. They also had a trial size that was much cheaper. https://www.thermofisher.com/order/catalog/product/78510

See attached. It may be useful

HI Rikki, I am planning to try ThermoFisher T-Per Tissue Protein Extraction Reagent in Pancreatic tissue. Have you ever tried it before? Also can you provide protocol for ThermoFisher T-Per Tissue Protein Extraction Reagent please.

Trizol is a very robust way to get both RNA and DNA, however, the DNA will be sheared especially if you homogenize vigorously. The protocol for Trizol claims that protein can be isolated from the organic phase but honestly it's a pain to actually get it.

how much protein lysis buffer should be used for a mice retina and how should keep it in lysis buffer

Hi Maryam,

The common rule is the ratio between 10-20 volumes of lysis buffer for 1 part(by weight) of tissue. Buffer should ideally contain protease inhibitors and to be ice-cold. Shaking on ice is good strategy for gentle protein extraction.

But protocol could vary depend of your needs.

So, first it is good idea to decide what you going to do with it and select right protocol based on your needs.

I wonder if anyone ever had an issue detecting proteins with a specific antibody, depending on the buffer used.

I have always made my RIPA buffer from scratch and everything was fine according to my controls. One day I decided to start ordering ready made RIPA buffer and things changed. We made several tests trying to figure this out comparing different sources of RIPA buffer and two antibodies that recognize the same target also from different companies.

If we prepare cell lysates in RIPA buffers from different companies and probe with the same antibody on the same membrane so I can compare the lysates, the results are totally different. I'm talking about going from no change to downregulation or the protein target. For one of the antibodies the result is the same for any buffer tested, but the other shows this problem.

Any input is appreciated.

Hi, Daniil

I have a Persian-language protocol. Would you like to send it to you?