Endoplasmic Reticulum (ER) proteins purification / isolation kit ER-Protein Capture Kit | Funakoshi

ER-Protein Capture Kit labels ER-associated proteins specifically and enables to isolate them by immunoprecipitation.
This kit is a powerful tool for global analysis of various ER proteins.

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Endoplasmic reticulum (ER) is the largest organelle in a cell and has unique and dynamic tubular or sheet structures.

ER plays essential roles in protein synthesis and carbohydrate metabolism, and is a traffic origin of secreted pathway proteins including the Golgi apparatus, exocytosis, plasma membrane and extracellular proteins.
In spite of the important roles of ER, biochemical isolation methods are highly limited presently because of complicated ER structures.
Some biochemical approaches with ultracentrifugation are utilized to roughly isolate ER membrane fractions, but conventional methods require time consuming procedures with specialized equipment and show frequent contamination of other organelles such as endosomes etc.
To access the function of ER proteins, an isolation method for ER associated proteins specifically with an easy procedure should be expected.

Our ER Protein Capture Kit is based on the ER localizable Reactive Molecule (ERM) technology originally developed by Dr. Itaru Hamachi and Dr. Tomonori Tamura, Kyoto University.
This kit enables to purify ER associated proteins to evaluate various ER roles with easy procedures and no special equipment such as ultracentrifuge machine.

A: ER-localizable Reactive Molecule (ERM)
B: Anti rhodol antibody

ERM is a small compound which has two units, a rhodol type green fluorescent dye and a thioester type protein labeling group.
The rhodol type dye has a high affinity to ER membranes and specifically and quickly accumulates into ER (Step-1: Left)
Immediately after the addition and specific accumulation of ERM into the ER, the labeling group of ERM reacts with nucleophilic amino acids in ER proteins forming a covalent bond to proteins to label the rhodol tag (Step-1: Right).
Subsequently cells are lysed by cell lysis buffers (Step-2), and rhodol tagged proteins are selectively purified by immunoaffinity purification with anti-rhodol antibody (Step-3). Purified rhodol tagged proteins are separated by SDS-PAGE (Step-4) and analyze d by LS/MS based proteomics or western blot method (Step-5) .

Hela cells were treated with ERM (100 nM) and organelle markers, Glibenclamide type ER staining, lysosomal staining, mitochondrial staining and Golgi apparatus staining dyes.
ERM was highly overlapped with ER (Piason coefficiency >0.9) but not correlated with the lysosome marker or mitochondria marker.
Only a small portion of staining by ERM was overlapped with the Golgi apparatus.
It was considered that this is attributed to the vesicle transport of ERM itself or rhodol labeled proteins from ER to the Golgi apparatus.

Excitation (blue) and fluorescent (red) spectrum.
Exmax /Emmax = 509/524 nm.
Commercial FITC filter sets are compatible.

HeLa cells in 10 cm dish were treated with 100 nM ERM for 1 hour and lysed by cell lysis buffer.
After total protein precipitation by chilled acetone, proteins were resolved in 4 % SDS lysis buffer (25 mM Tris (pH 7.6), 150 mM NaCl, 1% Nonidet P 40, 4% SDS, 1% sodium deoxycholate) with sonication, and then diluted 4 fold with RIPA buffer to ~1% SDS concentration.
Total protein concentration was measured with BCA assay and 2.5 mg of total proteins were applied for immunoaffinity purification.
Protein A-beads and anti-rhodol antibody complex was added to total protein solution and incubate for overnight at 4℃.
After incubation, the beads were washed and purified proteins were eluted with SDS-PAGE loading buffer.
Proteins were separated by SDS-PAGE and the gel was fixed.
After slicing the gel, sliced gels were subjected to in-gel digestion using trypsin/Lys-C.
The eluted peptides derived from the gel were purified and subjected to nanoLC-MS/MS analysis.
Total of 146 proteins were identified in this experiment. 63 proteins were categorized in ER resident proteins and 73 proteins were categorized in the secretory pathways such as membrane, Golgi apparatus, and extracellular proteins.
As secretory pathway proteins basically move to the final destination via ER, ERM based assay could identify ER associated proteins with around 93% probability.

HeLa cells were continuously grown in “light” SILAC media or “heavy” SILAC media.
The “heavy” isotope labeled cells and the “light” isotope labeled cells were treated with tunicamycin (2.5 μg/ml), a chemical inducer of ER stress, and DMSO as vehicle control for 4 hours, respectively.
After tunicamycin or DMSO treatment, both cells were incubated with 100 nM ERM for 1 hour and lysed by cell lysis buffer.
Equal amounts of “heavy” isotope labeled proteins and “light” isotope labeled proteins were mixed in a 1:1 ratio and rhodol labeled proteins were purified with anti-rhodol antibody /protein A beads and analyzed described above.
A total of 87 proteins were identified and quantified. Among 87 proteins, 39 proteins (45%) were classified as ER proteins and 84 proteins (97%) were assigned with ER associated proteins including secretory pathway proteins.
SILAC analysis indicates 6 proteins were upregulated by more than 2 fold in tunicamycin treated cells.
On the other hand, 7 proteins were downregulated by more than 2 fold in tunicamycin treated cells.

Endoplasmic Reticulum (ER) staining dye for long-term live imaging