PMID- 14654056
OWN - NLM
STAT- MEDLINE
DCOM- 20041012
LR  - 20190628
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 324
IP  - 1
DP  - 2004 Jan 1
TI  - Cross-linking approach to affinity capture of protein complexes from 
      chaotrope-solubilized cell lysates.
PG  - 137-42
AB  - Affinity capture methods are widely used for isolation and analysis of protein 
      complexes. Short peptide tags fused to the protein of interest normally 
      facilitate straightforward purification and detection of interacting proteins. We 
      investigated the suitability of applying C-terminally hexahistidine-tagged 
      interleukin-12 (IL-12) alpha- and beta-chains as "bait" proteins for cocapturing 
      novel binding partners using heterologous recombinant human embryonic kidney-293 
      (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from 
      cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity 
      resin under nondenaturing conditions. Retention of the alpha-chain on this matrix 
      was dependent on treatment of cell lysates with high concentrations of chaotropes 
      such as urea. Since under these conditions any noncovalent protein associations 
      are destroyed, prior cross-linking of proteins interacting with the alpha-chain 
      in intact cells was required. The use of the thiol-cleavable cross-linker 
      3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of 
      alpha-chain-binding proteins by means of dithiothreitol following purification. 
      Using this approach we were able to demonstrate a strong interaction between the 
      endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 alpha-chain that 
      was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay 
      presented here provides a simple approach to exposing concealed hexahistidine 
      tags while retaining native noncovalent protein interactions and should be 
      generally applicable in a range of pull-down or affinity capture methods aiming 
      at analysis of protein complexes.
FAU - Alloza, Iraide
AU  - Alloza I
AD  - Biomolecular Sciences Group, School of Pharmacy, Queen's University of Belfast, 
      97 Lisburn Road, Belfast BT9 7BL, UK.
FAU - Martens, Erik
AU  - Martens E
FAU - Hawthorne, Susan
AU  - Hawthorne S
FAU - Vandenbroeck, Koen
AU  - Vandenbroeck K
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Affinity Labels)
RN  - 0 (Calreticulin)
RN  - 0 (Cross-Linking Reagents)
RN  - 0 (Peptides)
RN  - 0 (Proteins)
RN  - 0 (Succinimides)
RN  - 0 (Sulfhydryl Compounds)
RN  - 187348-17-0 (Interleukin-12)
RN  - 4QD397987E (Histidine)
RN  - 7OV03QG267 (Nickel)
RN  - 8W8T17847W (Urea)
RN  - EVY5D0U6SH (dithiobis(succinimidylpropionate))
RN  - KA90006V9D (Nitrilotriacetic Acid)
RN  - T8ID5YZU6Y (Dithiothreitol)
SB  - IM
MH  - *Affinity Labels
MH  - Calreticulin/chemistry
MH  - Cell Line
MH  - *Cross-Linking Reagents
MH  - Dithiothreitol
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Histidine/chemistry
MH  - Humans
MH  - Interleukin-12/chemistry
MH  - Nickel/chemistry
MH  - Nitrilotriacetic Acid/chemistry
MH  - Peptides/chemistry
MH  - Plasmids
MH  - Protein Denaturation
MH  - Proteins/*chemistry
MH  - Succinimides/chemistry
MH  - Sulfhydryl Compounds
MH  - *Urea
EDAT- 2003/12/05 05:00
MHDA- 2004/10/13 09:00
CRDT- 2003/12/05 05:00
PHST- 2003/12/05 05:00 [pubmed]
PHST- 2004/10/13 09:00 [medline]
PHST- 2003/12/05 05:00 [entrez]
AID - S0003269703006249 [pii]
AID - 10.1016/j.ab.2003.09.017 [doi]
PST - ppublish
SO  - Anal Biochem. 2004 Jan 1;324(1):137-42. doi: 10.1016/j.ab.2003.09.017.