TITLE: Western blot, ELISA and enzymatic assays of reference proteins for subcellular fractionation 
AUTHORS: Saumel Perez Rodriguez, María  De Jesús Ramírez-Lira, Tune  Wulff, Bjørn  Gunnar Voldbor, Octavio  T Ramírez, Mauricio  A Trujillo-Roldán, Norma  A Valdez-Cruz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Subcellular fractionation of mammalian cells has been applied for the study of morphology, composition, structure and interactions between organelles, cellular and molecular biology and, more recently, the cell composition through omics approaches </div><div class = "text-block">⁠</div><div class = "text-block"><span>. Advantages of fractionation comprise but are not limited to obtaining fractions enriched in certain compartments for the study of cellular processes </span><span style = "font-style:italic;">in vitro</span><span style = "font-style:italic;"> </span></div><div class = "text-block">⁠</div><div class = "text-block">, locate and track proteins </div><div class = "text-block">⁠ and analyze post-translational modifications (PTM) of proteins processed along the secretory pathway </div><div class = "text-block">⁠ and protein composition of organelles</div><div class = "text-block"><span>⁠. Despite the existence of a wide variety of cell fractionation protocols and techniques, most of them are based on the identification or assignment of a certain isolated fraction to one or more subcellular compartments from the enrichment of this fraction in certain markers, the which are mostly protein. The quantification of these protein markers can be done through western blot, ELISA, enzymatic assays or proteomic studies. The present protocol is focused in the identification of proteins markers from several organelles, by WB, ELISA and enzymatic assays. The enrichment of isolated fractions in endoplasmic reticulum (ER), cytosol, nucleus, mitochondria, plasma membrane (PM), </span><span style = "font-style:italic;">cis</span><span>-Golgi and </span><span style = "font-style:italic;">trans</span><span>-Golgi can be assessed by detection of 78 kDa glucose-regulated protein (Grp78), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), histone H3, heat shock protein 60 (Hsp60), flotilin 1, golgin A5 and golgin-97, respectively, by WB, and also by ELISA in case of golgin-97. Enrichment of peroxisomes in fractions collected from sucrose gradients can be assessed by a catalase assay adapted from </span></div><div class = "text-block">. This protocol can be applied to any mammalian cell line during subcellular fractionation in order to quantify the enrichment of several organelles in the isolated fractions. </div></div>

[STEPS]
?. [Membrane preparation and protein transfer for WB assays]
Cut the PVDF membrane to the dimensions of the polyacrylamide gel from which the proteins are to be transferred.
?. [Membrane preparation and protein transfer for WB assays]
Soak the PVDF membrane in 100% methanol for , with a constant agitation.
?. [Membrane preparation and protein transfer for WB assays]
Discard methanol and soak the membrane in MilliQ water for , with a constant agitation.
?. [Membrane preparation and protein transfer for WB assays]
Discard water and soak the membrane in transfer buffer ( Tris,  glycine, 0.08% [w/v] SDS, 20% [v/v] methanol) for , with a constant agitation.
?. [Membrane preparation and protein transfer for WB assays]
After removing the polyacrilamide gel from the electrophoresis chamber, rinse it with MilliQ water 3 times to remove excess electrophoresis buffer salts and detergents, and soak it in transfer buffer for, with a constant agitation.
?. [Membrane preparation and protein transfer for WB assays]
Assemble the transfer cassette according to the reference manual of the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, CA, USA).
?. [Membrane preparation and protein transfer for WB assays]
Proceed to transfer at 20 V for 30-60 min according to the size of the protein to be detected and equipment performance.
?. [Membrane preparation and protein transfer for WB assays]
Wet the membrane in Ponceau staining (0.5% [w/v] Ponceau S, 1% [v/v] acetic acid), with constant agitation, until detection of protein bands.
Prior to WB assay, it should be corroborated that the protein transfer was successful. This step is optional but highly recommended to ensure that low or no detection of the protein of interest is due to its concentration in the experimental samples and not to poor transfer.
?. [Membrane preparation and protein transfer for WB assays]
Rinse the membrane with MilliQ water 3 times to eliminate excess Ponceau staining, and destain it with Tris-Glycine pH 8.3 ( Tris,  Glycine, 0.1% [w/v] SDS) until all previous staining has disappeared. Rinse the membrane with MilliQ water 3 times to eliminate excess detergent.
?. [Membrane preparation and protein transfer for WB assays]
Storage the membrane in phosphate buffer ( NaCl,  KCl,  Na2HPO4,  KH2PO4) at   until use.
4 °C
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Block the membrane in 5% (w/v) skimmed milk, 0.05% (v/v) Tween-20 in phosphate buffer  ( NaCl,  KCl,  Na2HPO4,  KH2PO4), for  at , with constant agitation.
0 Room temperature
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Incubate the membrane with Endoplasmic Reticulum Fraction Western Blot Cocktail (Abcam, Cambridge, MA, USA), diluted 2000 times in 0.5% (w/v) BSA, 0.05% (v/v) Tween-20 in phosphate buffer for  at , with constant agitation.
0 Room temperature
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Wash the membrane 3 times in 0.05% (v/v) Tween-20 in phosphate buffer, for  in each wash, with constant agitation.
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Repeat step 12.
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Incubate the membrane with horseradish peroxidase (HRP) Conjugated Secondary Antibody Cocktail (Abcam, Cambridge, MA, USA), diluted 2500-fold in 0.5% (w/v) BSA, 0.05% (v/v) Tween-20 in phosphate buffer,  for  at , with constant agitation.
0 Room temperature
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Repeat step 12.
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Wash the membrane in phosphate buffer for  , and wet it in a 1:1 (v/v) mix of Luminol/Enhancer Solution and Stable Peroxide Solution from SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).
?. [Detection of Grp78, Gapdh and histone H3 by WB ]
Acquire the WB image in a LI-COR C-DiGit Chemiluminescence Western Blot Scanner by using Image Studio software in high sensitivity mode (LI-COR Biosciences, Lincoln, NE, USA).
Alternatively, the resulting bands can be visualized by X-ray films or other CCD camera–based digital imaging instruments.
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Block the membrane in 3% skimmed milk, 0.1% (v/v) Tween-20 in Tris-buffered saline (TBS,  NaCl,  Tris-HCl, ) for  at , with constant agitation.
0 Room temperature
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Incubate the membrane with the corresponding primary antibody, diluted in 3% skimmed milk, 0.1% (v/v) Tween-20 in TBS,  at , with constant agitation.
4 °C
Anti golgin-97 (GTX114445, GeneTex, CA, USA), anti flotilin 1 (GTX104769, GeneTex, CA, USA), anti Hsp60 (GTX110089, GeneTex, CA, USA) and anti golgin A5 (GTX104255, GeneTex, CA, USA) antibodies are diluted 2 000,  2 000, 10 000 and 2 000 times, respectively.
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Wash the membrane 3 times in 0.1% (v/v) Tween-20 in TBS for  in each wash, with constant agitation.
In the cases of anti Hsp60 and anti golgin A5 antibodies, the time of each wash should be extended to .
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Incubate the membrane with an anti-rabbit IgG conjugated to HRP,  used as secondary antibody and diluted 2000 times in 3% skimmed milk, 0.1% (v/v) Tween-20 in TBS, for  at , with constant agitation.
0 Room temperature
Any other anti-rabbit secondary antibody, conjugated to the HRP enzyme, may be used at the manufacturer's recommended dilution.
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Repeat step 21.
?. [Detection of golgin-97, golgin A5, flotilin 1 and HSP60 by WB ]
Repeat steps 17-18.
?. [Detection of golgin-97 by ELISA]
Coat ELISA high binding plates (Greiner Bio-One GmbH, Austria) with  of E. coli ATCC 53606 and CRL-12444 cell homogenates, and samples from differential centrifugation, all diluted in a final volume of  of sodium carbonate-bicarbonate buffer, , for  at.
4 µg
200 µl
4 °C
Homogenates from E. coli and CRL-12444 cells are used as negative and positive controls of the assay, respectively. E. coli ATCC 53606 can be replaced by any other E. coli strain, and CRL-12444 by any other CHO cell line as well.
To obtain E. coli and CHO cell homogenates, centrifuge CHO cells at  , and E. coli cells at  , wash cell pellets twice in phosphate buffer  (  NaCl,   KCl,   Na2HPO4,   KH2PO4) and lyse them by solubilization in isoelectric focusing buffer (IEF,   urea,   thiourea, 2% [w/v] CHAPS,   dithiothreitol) supplemented with 10% (v/v) of SigmaFast Protease Inhibitor Cocktail. Sonicate the lysates twice for   at 10 µm, and centrifuge at  . Storage at    until use.
?. [Detection of golgin-97 by ELISA]
Block the plates with  of 1% (w/v) BSA and 0.05% Tween-20 in phosphate buffer, for  at .
200 µl
4 Room temperature
?. [Detection of golgin-97 by ELISA]
Wash the plates 5 times with per well of 0.05% (v/v) Tween-20 in phosphate buffer  ( NaCl,  KCl,  Na2HPO4,  KH2PO4) at .
200 µl
4 Room temperature
?. [Detection of golgin-97 by ELISA]
Incubate the plates with anti golgin-97 antibody (GTX114445, GeneTex, CA, USA), diluted 2000-fold in 1% (w/v) BSA and 0.05% Tween-20 in phosphate buffer, for  at , with per well.
4 Room temperature
100 µl
?. [Detection of golgin-97 by ELISA]
Repeat step 26.
?. [Detection of golgin-97 by ELISA]
Repeat step 26.
?. [Detection of golgin-97 by ELISA]
Incubate the plates with an anti-rabbit IgG conjugated to HRP, used as secondary antibody and diluted 1000 times in 1% (w/v) BSA and 0.05% Tween-20 in phosphate buffer, and incubate for  at  with  per well.
4 Room temperature
100 µl
?. [Detection of golgin-97 by ELISA]
Add  per well of SigmaFast o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), prepared according to manufacturer’s recommendations, and incubate for  at.
100 µl
4 Room temperature
Other HRP substrates used for ELISA can be used as well.
?. [Detection of golgin-97 by ELISA]
Stop the enzymatic reaction by addition of  of HCl 10% (v/v) per well.
50 µl
?. [Detection of golgin-97 by ELISA]
Read the absorbance at 490 nm.
?. [Enzymatic assay of catalase]
Mix  of each fraction with  of  phosphate buffer ( KH2PO4,  K2HPO4), , and  of 1% (v/v) Triton X-100, and place in a 12x75 mm glass test tube.
20 µl
30 µl
50 µl
This assay has been optimized to measure catalase activity in fractions collected from sucrose gradients. The assay was adapted from .
?. [Enzymatic assay of catalase]
Add  of 30% (v/v) hydrogen peroxide to each tube and mix.
50 µl
?. [Enzymatic assay of catalase]
After  incubation, measure the height of foam column.
?. [Enzymatic assay of catalase]
Calculate the specific activity of catalase (mm/mg) as the ratio between foam height (mm) and the protein quantity added to the tube (mg).
?. [Detection of golgin-97 by ELISA]
Repeat step 26.