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Nondenaturing Protein Immunoprecipitation from Mammalian
Cells
William P. Tansey
ABSTRACT
This protocol describes a nondenaturing immunoprecipitation (IP) for mammalian cells. Where
possible, we prefer to use denaturing IPs to recover labeled proteins from pulse-chase
experiments. However, the nondenaturing protocol is useful when one wishes to separate
soluble from insoluble proteins, or when the antibody being used recognizes a native
epitope.
Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Buffers,
Solutions, and Reagents |
|
|
|
| Antibody of choice |
Visit the Antibody Explorer for all your antibody needs. |
| Laemmli (SDS-PAGE) sample buffer (2X) |
S3401 |
Sample Buffer, Laemmli 2× Concentrate |
|
| Protein A Sepharose (50%, pre-swollen) |
P3391 |
Protein A-Sepharose® from Staphylococcus aureus |
|
| Protein G Sepharose (50%, pre-swollen) |
P3296 |
Protein G Sepharose®, Fast Flow, recombinant, expressed in
Escherichia coli, aqueous ethanol suspension |
|
| NaCl |
S3014 |
Sodium chloride, for molecular biology |
|
| Pefabloc |
76307 |
Pefabloc® SC |
|
| NP-40 |
NP40S |
Tergitol® solution, Type NP-40, 70% in H2O |
|
| Deoxycholate (DOC) |
D6875 |
21-Hydroxyprogesterone |
|
| SDS (10%) |
L4390 |
Sodium dodecyl sulfate, for molecular biology |
|
| Tris-Cl |
T5941 |
Trizma® hydrochloride, for molecular biology, Biotechnology
Performance Certified |
|
| Dithiothreitol (DTT) |
D9779 |
DL-Dithiothreitol, for molecular biology |
|
| Bromophenol blue |
B0126 |
Bromophenol Blue |
|
| Aprotinin |
A6103 |
Aprotinin from bovine lung, recombinant, expressed in
Nicotiana |
|
| Pepstatin |
P5318 |
Pepstatin A |
|
| Leupeptin |
L9783 |
Leupeptin hydrochloride |
|
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The Sigma-Aldrich products listed for this specific protocol were selected either
to match or to supplement the products listed within the actual protocol. The
products/reagents from Sigma-Aldrich have been qualified for usage, but may not
have been validated for this specific application. Please refer to the detailed
product description on the usage of specific products of interest.
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