Protein Extraction Reagents

Cell Lysis Buffer M

This product is a reagent for extracting cytoplasmic proteins from animal cells.

RIPA Buffer

This product is a reagent for extracting cytoplasmic, nuclear, and cell membrane proteins from animal cells. Certain membrane proteins cannot be extracted.

LysoPure™ Nuclear and Cytoplasmic Extractor Kit

This product is a kit for fractionating and extracting cytoplasmic, soluble nuclear, and insoluble nuclear proteins from cultured cells and tissues. After extraction of cytoplasmic fractions and soluble nuclear proteins, insoluble nuclear fraction proteins can be solubilized with SDS Lysis Buffer and extracted as denatured proteins that can be analyzed by Western blotting, etc.

Features

• Cytoplasmic and nuclear proteins can be fractionated and extracted in a stepwise manner.
• The entire process takes only approximately 2 hours.
• The nuclear fraction is less cross-contaminated with cytoplasmic proteins.
• The nuclear fraction can be extracted separately into soluble and insoluble fractions.

Protocol

Kit composition (for 100 times use)

• Nuclear Fractionation Buffer・・・100 mL
• Nuclear Extraction Buffer・・・75 mL
• SDS Lysis Buffer・・・20 mL

* The number of uses is based on a cell number of 1 x 10⁷/use.

Volume of reagents required for fractionation

• Cell number (cells)	Nuclear Fraction buffer (μL)	Nuclear Extraction buffer (μL)	SDS Lysis buffer (μL)
  2 x 106	100	50	50
  5 x 106	250	125	100
  1 x 107	500	250	200

• Tissue amount (mg)	Nuclear Fraction buffer (μL)	Nuclear Extraction buffer (μL)	SDS Lysis buffer (μL)
  20	100	50	100
  50	500	250	200
  100	1,000	500	400

Note: In addition, 500 μL each of nuclear fractionation buffer and nuclear extraction buffer are used in the washing process.

Performance comparison-Western blotting

Experimental conditions

Sample amount at the start

Hela：1 x 10⁷cells
K562：1 x 10⁷cells
Mouse Liver：100 mg

Loading amount

10 μg/Lane

Electrophoresis gel

SuperSep™ Ace, 5-20%

Running buffer

SDS-PAGE Buffer, pH8.5

Molecular weight marker

WIDE-VIEW™ Prestained Protein-Size Marker III

Luminescent reagent

ImmunoStar^® Zeta

Antibodies (primary and secondary)

Primary antibody(dilution factor)	Secondary antibody(dilution factor)	Molecular Weight (kDa)
α-tublin (1:3,000)	Mouse (1:10,000)	50
Histon H1 (1:2,000)		32-33
Histon H3 (1:2,000)	Rabbit (10,000)	17
Nup98 (1:1,000)		98
Lamin B1 (1:3,000)		70
COX IV (1:2,000)		17