IECs were isolated by sequential incubation of intestinal tissue in 1 mM dithiothreitol (DTT) and 1.5 mM EDTA solutions as described previously40 (link). Cell lysates from IECs and keratinocytes were prepared as described41 (link),42 (link). Lysis buffer was supplemented with protease and phosphatase inhibitor tablets (Roche). Cell lysates were separated on SDS–PAGE and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were probed with primary antibodies against the following proteins: RIPK1 (610459, BD Biosciences), RIPK3 (ADI-905-242-100), cIAP1 (ALX-803-335-C100, Enzo), IκBα (sc-371), TRAF2 (sc-876), TRAF3 (sc-949), TRADD (sc-7868), p65 (sc-372), c-Rel (sc-71),HDAC1 (sc-7872), β-actin, (sc-1616, Santacruz), phospho-p65 (3033), JNK (9252), RelB (4922), phospho-p38 (9211), p38 (8690), phospho-ERK (9101), ERK (9102, Cell Signaling), phospho-JNK (44-682G, Invitrogen), p100/52 (NR1495), p105/50 (NR1157, NCI BRB), α-tubulin (T6074, Sigma), TRAF6 (597, MBL), c-FLIP (AG-20B-0005-C100, Adipogen), followed by secondary HRP-coupled antibodies (GE Healthcare and Jackson ImmuneResearch) and developed with chemiluminescent detection substrate (GE Healthcare and Thermo Scientific).
Protocol detail
Isolation and Analysis of Intestinal Epithelial Cells
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
protease and phosphatase inhibitor tablets
IPVH00010
RIPK1
RIPK3
cIAP1
ALX-803-335-C100
β-actin
phospho-JNK
chemiluminescent detection substrate
Actin
Antibodies
Buffer
C rel
Cell
Ciap1
Dithiothreitol
Edta
Intestinal
Iκbα
Keratinocytes
Membranes
P100
Phosphatase
Protease
Proteins
Pvdf
Ripk1
Ripk3
Sc 372
Sds page
Tissue
Traf2
Traf3
Traf6
α tubulin
Corresponding Organization :
Other organizations : University of Cologne, University of Massachusetts Chan Medical School, Universität Ulm, Medizinische Hochschule Hannover
Top 5 similar protocols
Protocol cited in 1 other protocol
Variable analysis
independent variables
- Sequential incubation of intestinal tissue in 1 mM dithiothreitol (DTT) and 1.5 mM EDTA solutions
- Levels/expression of the following proteins: RIPK1, RIPK3, cIAP1, IκBα, TRAF2, TRAF3, TRADD, p65, c-Rel, HDAC1, β-actin, phospho-p65, JNK, RelB, phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, p100/52, p105/50, α-tubulin, TRAF6, c-FLIP
- Lysis buffer supplemented with protease and phosphatase inhibitor tablets (Roche)
- Cell lysates separated on SDS–PAGE and transferred to PVDF membranes
- Not explicitly mentioned
- Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!