Thermo Scientific Pierce RIPA and IP Lysis Buffers

Thermo Scientific Pierce RIPA Buffer Compatibility with protease inhibitors prevents proteolysis.

Highlights:•Convenient – ready-to-use solution• Flexible – compatible with many applications, including reporter

assays, protein assays, immunoassays and protein purification• Versatile – enables extraction of cytoplasmic, membrane and

nuclear proteins

Excellent protein yieldTwo different ratios of Pierce RIPA Buffer volume to number of HeLa cells were analyzed for total protein yield; 1.25 x 106 and 2.5 x 106 cells were lysed with 1 and 0.5mL, respectively, of Pierce and Supplier S RIPA Formulations. Equal amount of proteins were obtained using either formulation when 1.25 x 106 cells were lysed with 1mL of Buffer (Figure 1A); however, protein yield was significantly greater using Pierce RIPA Buffer when 2.5 x 106 cells were lysed with 0.5mL of buffer (Figure 1B).

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Lysis: 1.25 x 106 cellswith 1mL of Buffer

Lysis: 2.5 x 106 cellswith 0.5mL of Buffer

A.Thermo Scientific

PierceSupplier S

hsp90(Cytosolic)

Nucleoporin(Nuclear)

H: HeLa cellsA: A431 cells

Flotillin(Membrane)

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Free sample available!

The Thermo Scientific Pierce RIPA Buffer is a reliable buffer used to lyse cultured mammalian cells from both plated cells and cells pelleted from suspension cultures. It enables the ex-traction of membrane, nuclear and cytoplasmic proteins and is compatible with many applications, including reporter assays, the Pierce BCA Protein Assay, immunoassays and protein puri-fication. Inhibitors such as the Thermo Scientific Halt Protease Inhibitor Cocktail (Product # 78425) and Halt Phosphatase Inhibitor Cocktail (Product # 78420) are also compatible with the Pierce RIPA Buffer and can be added before use to prevent proteolysis and maintain protein phosphorylation.

To request your free 25mL sample (Product # 89900S), visit www.thermoscientific.com/perbio.Offer void where prohibited. Offer valid from March 1, 2011–August 31, 2011, or while supplies last. Limit one free sample per laboratory.

Figure1.TheThermoScientificPierceRIPABufferextractsequivalentormoreproteinthantheSupplierSRIPABuffer. Pierce and Supplier S RIPA Buffer at volumes of 1 and 0.5mL were added separately to 1.25 x 106 and 2.5 x 106 HeLa cells, respectively. Cells were thoroughly resuspended and incubated for 10-15 minutes on ice with occasional swirling of tubes. After clarification of cell lysates by centrifu-gation, protein extraction was determined using the Pierce BCA Protein Assay Kit (Product # 23225). Protein extraction was equal when 1.25 x 106 cells were lysed with 1mL of buffer (Figure 1A), but the Pierce RIPA Buffer extracted more protein than Supplier S’s buffer when 2.5 x 106 (Figure 1B) cells were lysed with only 0.5mL.

Figure2.Isolationofmembrane,nuclearandcytosolicproteinsusingThermoScientificPierceRIPABuffer.The Pierce RIPA Buffer extracts proteins from (A) membrane, (B) nuclear and (C) cytosolic fractions. The Pierce RIPA Buffer was supplemented with the Halt Protease Inhibitor Cocktail (Product # 78410) and used to lyse HeLa and A431 cells. Western blotting was performed using mouse anti-flotillin, -nucleoporin and -hsp90 antibodies (BD Biosciences) at 0.25µg/mL, 1µg/mL and 0.25µg/mL, respectively, and goat anti-mouse-HRP (20ng/mL, Product # 31430). The signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Product # 34080).

OrderingInformationProduct#

Description

Pkg.Size

89900 PierceRIPABuffer 100mL

89901 PierceRIPABuffer 250mL

Please see the reverse side for information on the Pierce IP Lysis Buffer.

Proteins (7µg) obtained from HeLa or A431 cells were separated on a 4-12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and probed for flotillin (membrane), nucleoporin (nuclear) and hsp90 (cytosolic) proteins and detected by chemiluminescence using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Product # 34080). Proteins from all the three different compartments of the cell were readily detected in cell lysates prepared using Pierce RIPA Buffer (Figure 2).

Thermo Scientific IP Lysis Buffers

Thermo Scientific Pierce IP Lysis BufferThe Thermo Scientific Pierce IP Lysis Buffer is a mammalian whole cell lysis buffer based on a modified RIPA buffer formulation without SDS. This moderate-strength lysis buffer is able to effectively solubilize cellular proteins (Figure 1) but does not liberate genomic DNA or disrupt protein complexes like RIPA buffer does. The Pierce IP Lysis Buffer is especially formulated for pull-down and immunoprecipitation assays and as a wash buffer for affinity resins (Figure 2); however, it is also compatible with many other applications, including the Thermo Scientific Pierce BCA and 660nm Protein Assays, protein purifi-cation and immunoassays (e.g., ELISA, Western blot).

Highlights:• Optimized for immunoprecipitation and pull-down assays• Compatible with protein, reporter and immunoassay procedures• Ready-made formula is effective for extracting cytoplasmic,

membrane and nuclear proteins• Gentle formulation helps maintain protein complexes• Does not liberate genomic DNA, which can cause high

sample viscosity

The Pierce IP Lysis Buffer is composed of Tris•HCl, NaCl, EDTA, NP-40 and glycerol. Although this buffer does not contain protease or phosphatase inhibitors, the Thermo Scientific Halt Protease Inhibitor and Phosphatase Inhibitor Cocktails are compatible and can be added just before use to prevent proteolysis and maintain protein phosphorylation.

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Sigma CelLytic M Thermo Scientific PierceIP Lysis Buffer

RIPA Buffer0.00

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1

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3.5

Thermo Scientific Pierce IP Lysis Buffer

L E L E

RIPA Buffer

PP2A

EGFR

Please see the reverse side for information on the Pierce RIPA Buffer.

© 2011 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Prices listed herein were accurate at the time of printing. CelLytic is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Facebook is a registered trademark of Facebook, Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

Free sample available!

www.thermoscientific.com/perbio

OrderingInformation

Product# Description Pkg.Size87787 PierceIPLysisBuffer 100mL

87788 PierceIPLysisBuffer 250mL

Figure1.Themoderate-strengthThermoScientificPierceIPLysisBufferproduceshighyield. A549 cells (2 x 106) were lysed using 200μL of three different lysis buffers, clarified by centrifugation and measured for protein concentration using the Pierce BCA Protein Assay (Product # 23227).

Figure2.TheThermoScientificPierceIPLysisBufferenableshighlyefficientimmunoprecipitations.A549 cell lysates (200μg) were generated using different lysis buffers. EGFR and PP2A were immunoprecipitated using the Thermo Scientific Pierce Crosslink IP Kit (Product # 26147) and anti-EGFR antibody (Stressgen) or anti-PP2A antibody (Millipore). The immunoprecipitations were washed with the same lysis buffer used to lyse the cells. Lysates representing 5% of the total load (L) and elution (E) fractions were blotted with anti-EFGR or anti-PP2A antibody and using Thermo Scientific SuperSignal West Dura Substrate (Product # 34076) for detection.

To request your free 25mL sample (Product # 87787S), visit www.thermoscientific.com/perbio. Offer void where prohibited. Offer valid from March 1, 2011–August 31, 2011, or while supplies last. Limit one free sample per laboratory.

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