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1. Potential Problems of Protein
Extraction Using RIPA Buffer
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2. Staring Materials for
Protein Extraction
Animal Cultured
Cells/Tissues
Plants
Microorganisms
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3. Methods For Cell/Tissue Lysis
•Physical
Sonication, homogenization, grinding, heating, Freeze and thaw, low osmosis pressure
etc.
• Chemical
High salt, extreme basic or acid solutions
Surfactants ----Denaturing (SDS), non-denaturing (NP-40)
Acid labile (Proteus) and acid non-labile
Strong reducing agents (urea), Guanidine-HCl
• Biological
Virus (phages), Enzymes (lysozme. Zyomlase and lyticase etc)
Combination of Physical, Chemical, Biological Methods

4. About RIPA Buffer
•RIPA buffer (Radioimmunoprecipitation Assay buffer)
has been widely used for protein extraction from animal
cells/tissues
2. It has been a “Gold standard” for total protein
extraction for a long time
3. RIPA buffer has been used in a variety of qualitative
and quantitative experiment, such as WB, IP and
ELISA.
4. There are many variations for RIPA buffer but many
use classical one

5. RIPA Buffer Compositions
Classical RIPA buffer Modified RIPA Buffer
Tris-HC 50 mM Tris-HC 50 mM
NaCl 150 mM NaCl 150 mM
1% Triton X-100 1% NP-40
1% Na-deoxycholate 1% Na-deoxycholate
0.1% SDS
1 mM EDTA 1 mM EDTA

6. Questions About RIPA Buffer
• Is the protein profile complete?
• Is there any side by side comparison
with other methods?
• What is the potential problems of
using RIPA buffer for protein
extraction?

7. Methods:
1. Cell lysates were prepared from un-activated (Lanes 1 and 5)
and activated (Lanes 2-4 and 6-8) T lymphocytes by RIPA
buffer and 2% SDS + sonication
2. WB was performed using anti-caspase -3, Caspase-7, Parp and
GD4
Major Findings:
RIPA buffer artificially activates caspases in activated T
lymphocytes
Conclusion: Be careful about data interpretation when RIPA
buffer is used for apoptosis studies
RIPA Buffer Artificially Activate Caspases

8. RIPA Buffer Artificially Activate Caspases
(Zapata et al., (1998). J Biol Chem 1998, 273:6916-6920.)

9. RIPA Buffer Artificially Increase Kinase Activity
(Deseau et al., (1987). J Cellular Biochem 1987, 35:113-128).
Methods:
•Cultured colon cancer cells were lysed by
RIPA buffer and NP-40
•Immunoprecipitations were performed with
specific Mab
•Same amounts of precipitated pp60c-src
were used for kinase assays

10. RIPA Buffer Artificially Increase Kinase Activity
(Deseau et al., (1987). J Cellular Biochem 1987, 35:113-128).

11. Major Findings
1.The kinase activity was seven folds
higher with RIPA than NP-40.
2. The increased activity is an artifact
induced by RIPA buffer
Conclusion: Kinase activity assays using
RIPA buffer should be interpreted with
caution

12. Comparison of Phosphorylation Patterns of RIPA and
Spin-Column Extracted Proteins
(Courtesy of G. Szanda,NIH/NIAAA/LPS)
Methods:
•Chemically induced and non-induced human cell
lines were used in the research
•Total proteins were extracted from above samples
•The proteins were separated in SDS-PAGE and
probed with antibodies
•Patterns of protein phosphorylation is compared
side by side

13. Comparison of Phosphorylation Patterns of RIPA and Spin-
Column Extracted Proteins
(Courtesy of G. Szanda,NIH/NIAAA/LPS)

14. Comparison of Phosphorylation Patterns of RIPA and Spin-
Column Based Protein Extraction
(Courtesy of G. Szanda,NIH/NIAAA/LPS)
Conclusions:
•Protein samples are equally loaded as evidenced by actin
bands intensity
•State3 shows the same pattern for both extraction methods
•Protein 56 shows significant difference between the two
methods
•Spin-column extracted protein shows anticipated results but
RIPA-extracted protein shows the opposite results

15. Comparison of RIPA Extracted Proteins By
MS Analysis
Lamber CM Ngoka (2008). Proteome Science. 6:1 (1).
Methods:
•Breast Cancer Tissues were extracted by RIPA
buffer
•Post-RIPA buffer extraction insoluble fractions
were extracted by urea buffer
•The protein profiles were compared using MS
analysis

16. Comparison of RIPA Extracted Proteins By MS
Analysis
Lamber CM Ngoka (2008). Proteome Science. 6:1 (1).

17. Conclusions
1. Post-RIPA buffer Extraction fractions contain a variety
of Proteins
2. Nearly All Extracellular Matrix Proteins are found
exclusively in the Insoluble fraction
3. The molecular weight of protein found in RIPA-
insoluble fraction is in average 60% higher
4. The samples should be extracted by RIPA and urea
buffer to avoid biased results

18. Protein Profiles of RIPA Extracted and Spin
Column Extracted Proteins By SDS-PAGE
BioTechniques 61:272-275 (December 2016)
Methods:
• Proteins were extracted from mouse samples by
Spin-column based method and RIPA buffer
•Insoluble fraction of post RIPA extraction fractions
were further extracted by spin-column based method
•Extracted proteins were separated by SDS-PAGE and
banding patterns compared by Commassie blue
staining.

20. 1. Protein lost to pellet ranging from >120 KD to
<20 KD.
2. The most obvious is low molecular weight
proteins
3. Protein lose is selective and non-
proportional
4. The profiles of lost proteins vary from sample
to sample
Major Findings

21. Summary
1. The protein profiles extracted by RIPA buffer is incomplete.
Artifacts have been reported.
2. Many proteins are lost to the insoluble fraction
3. Almost all high molecular weight ECM proteins are lost as
evidenced by MS analysis
4. Low molecular weight proteins are also lost to the pellet
5. Caution must be taken for data interpretation using RIPA
buffer only

22. Contact Us
Invent Biotechnologies
Address: 1864 Berkshire Ln N, Plymouth, MN
55441, USA
Phone No : 952-400-6898
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