High Quality RIPA Lysis Buffer(Strong) for Tissue and Cell (with PMSF)

Product Introduction

Product Information：

Yanbiotech's Plant RIPA Lysis Buffer is a traditional rapid lysis buffer for plant cells, tissues, or protoplasts. The protein samples obtained using Plant RIPA Lysis Buffer can be used for routine PAGE, Western Blot, Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), ELISA, etc.

This product can be used for plant cell, tissue, or protoplast samples, as well as for animal cell or tissue samples, fungal, or bacterial samples.

RIPA originally stands for Radio Immunoprecipitation Assay.

The main active ingredients of Plant RIPA Lysis Buffer (Strong) include 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS (detergents), as well as various inhibitors like sodium orthovanadate, sodium fluoride, EDTA, and leupeptin. It can effectively inhibit protein degradation.

The protein concentration of samples lysed with Plant RIPA Lysis Buffer can be determined using a BCA Protein Assay Kit. Due to the high concentration of detergents, the Bradford method should not be used to determine the protein concentration of samples lysed with this buffer.

Storage Conditions：

-20ºC ，12 months.

Precautions：

For optimal performance, avoid excessive freeze-thaw cycles. It is recommended to aliquot the product after thawing for single use.

PMSF must be prepared by the user. Alternatively, you can purchase the Protease & Phosphatase Inhibitor Cocktail (General Use, 50X) for overall better effect, or select the appropriate inhibitor mixture based on specific applications: Protease & Phosphatase Inhibitor Cocktail (For Plant Sample Extraction, 50X), Protease & Phosphatase Inhibitor Cocktail (General Use, MS Compatible, 50X), Protease & Phosphatase Inhibitor Cocktail (For Mammalian Sample Extraction, 50X), Protease & Phosphatase Inhibitor Cocktail (For Fungal or Yeast Extraction, 50X), Protease & Phosphatase Inhibitor Cocktail (For Bacterial Extraction, 50X). If detection of phosphorylated proteins is not required, a protease inhibitor cocktail without phosphatase inhibitors can also be selected.

All steps for lysing samples should be performed on ice or at 4°C.

This product is intended for research use by qualified professionals only. Not for use in clinical diagnosis or treatment. Not for food or drug use. Do not store in ordinary residences.

For your safety and health, please wear a lab coat and disposable gloves during operation.

Instructions for Use:

1. For Cultured Plant Cell Samples:

a. Thaw the Plant RIPA Lysis Buffer and mix well. Shortly before use, add PMSF to the appropriate amount of lysis buffer to a final concentration of 1mM, or add the suitable Protease & Phosphatase Inhibitor Cocktail as required by the experiment.

b. Centrifuge to harvest plant cells. Add 100-200 µl of lysis buffer per 0.5-1 million cells. Pipette several times to ensure thorough contact between the lysis buffer and cells. Lyse on ice for 2-10 minutes

c. After complete lysis, centrifuge at 10,000-14,000 g for 3-5 minutes. Collect the supernatant for subsequent PAGE, Western Blot, immunoprecipitation, etc.

2. For Plant Protoplast Samples:

a. Mince the tissue into small pieces and prepare protoplasts.

b. (Optional) Transform protoplasts with plasmid, continue culturing for 16-48 hours, and apply appropriate experimental treatments as needed.

c. Collect protoplasts by low-speed centrifugation at 100-500 g.

d. Thaw the Plant RIPA Lysis Buffer and mix well. Shortly before use, add PMSF to the appropriate amount of lysis buffer to a final concentration of 1mM, or add the suitable Protease & Phosphatase Inhibitor Cocktail.

e. Add 100-200 µl of lysis buffer per 0.5-1 million protoplasts. Gently flick the tube bottom to lyse the cells thoroughly. After complete lysis, no significant protoplast pellet should remain. If the protoplast quantity is large, it must be aliquoted into tubes containing 0.5-1 million protoplasts each before lysis. Large clumps of 0.5-1 million protoplasts are difficult to lyse completely, while smaller amounts (0.5-1 million) are relatively easier to lyse thoroughly as the lysis buffer can contact them more readily.

3.  For Plant Tissue Samples：

a. Mince the plant tissue into small fragments.。

b. Thaw the Plant RIPA Lysis Buffer (Strong) and mix well. Shortly before use, add PMSF to the appropriate amount of lysis buffer to a final concentration of 1mM, or add the suitable Protease & Phosphatase Inhibitor Cocktail.

c.  Add 100-200 µl of lysis buffer per 20 mg of plant tissue. (If lysis is insufficient, more lysis buffer can be added appropriately. If a higher protein concentration is needed, the amount of lysis buffer can be reduced accordingly.)

d. Homogenize using a glass homogenizer or a handheld tissue grinder until completely lysed. Alternatively, the tissue sample can be frozen, ground in liquid nitrogen, and then the lysis buffer can be added after thorough grinding.

e. After complete lysis, centrifuge at 10,000-14,000 g for 3-5 minutes. Collect the supernatant for subsequent PAGE, Western Blot, immunoprecipitation, etc.

f. If the plant tissue itself is very fine and tender, it can be appropriately sheared and then directly added to the lysis buffer. Perform vigorous vortexing to lyse the sample thoroughly. Then centrifuge similarly to collect the supernatant for subsequent experiments. The advantage of direct lysis is convenience, eliminating the need for homogenizers or grinding equipment. The disadvantage is that lysis may not be as thorough as with homogenization or grinding.

Note: A small transparent gel-like mass often appears in the lysate from Plant RIPA Lysis Buffer, which is a normal phenomenon. This transparent gel is a complex containing genomic DNA, etc. When detecting proteins not tightly bound to genomic DNA, the supernatant can be directly taken for subsequent experiments after centrifugation. If detecting proteins that are very tightly bound to the genome, this transparent gel can be disrupted and dispersed by sonication, followed by centrifugation to collect the supernatant. For detecting common transcription factors, sonication is usually not necessary.