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Santa Cruz discontinued a large number of its polyclonal products as a result of the USDA settlement that was made public May 19th 2016

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20. Make buffer fresh just prior to use.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised October 2016

Cell Fractionation Kit Protocol

A. Buffers

  • Cytoplasm Isolation Buffer (CIB) – 10 ml, Store at -20°C.
  • Membrane Isolation Buffer (MIB) – 10 ml, Store at -20°C.
  • Cytoskeleton/Nucleus Isolation Buffer (CyNIB) – 5 ml, Store at -20°C.
  • Protease Inhibitor Cocktail (100X) (#5871) – 250 µl, Store at 4°C.

B. Notes

  • All steps, except for the addition and sonication of the CyNIB buffer, should be done on ice or at 4°C.
  • Adherent or suspension cultured cells can be used for this assay.
  • 1X protease inhibitors (#5871) (5 μl of 100X per 500 μl buffer, included in kit) and 1 mM fresh PMSF (#8553) (2.5 μl of 200 mM PMSF per 500 μl buffer, not included in kit) should be added to each buffer immediately before use.
  • Phosphatase inhibitors are already included in the buffers. There is no need to add them.
  • Please refer to Table 1 for the appropriate volumes for your specific cell concentration.
  • The volumes given in Sections D and E are based on cell counts obtained from HeLa cells at ~90% confluency in a 10 cm cell culture dish (5 x 106 cells).
  • If the CyNIB buffer is cloudy after thawing, please warm the solution in a 37°C water bath until the solution is clear.
  • Be cautious when saving fractions so that you do not get any contamination from the resulting pellet.
  • All lysates should be stored at -20°C for short term storage (less than 1 month) or -80°C for long term storage (greater than 1 month).

C. Isolating Cell Population

For adherent cells

  1. Wash plate with cold 1X PBS.
  2. Trypsinize the plate.
  3. Add cold growth media to deactivate trypsin.

For both adherent and suspension cells

  1. Spin down cells at 350 x g for 5 min.
  2. Aspirate media.
  3. Wash cell pellet with cold 1X PBS.
  4. Resuspend pellet in 0.5 ml of cold 1X PBS.
  5. Count live cells using Trypan Blue and a hemacytometer.

D. Detection of Proteins

  1. Aliquot 100 µl of cell suspension into a 1.5 ml tube for the whole cell lysate (WCL).
  2. Add 60 μl of 3X SDS Loading Buffer with DTT (#7722) to make a final volume of 160 µl of WCL.
  3. Sonicate WCL tube for 15 sec at 20% power 3 times, heat for 5 min at 95°C, and centrifuge for 3 min at 15,000 x g.

E. Cell Fractionation

  1. Aliquot the remaining 400 µl into a 1.5 ml tube.
  2. Centrifuge for 5 min at 500 x g at 4°C.
  3. Aspirate the supernatant.
  4. Resuspend pellet in 500 μl of CIB.
  5. Vortex for 5 sec.
  6. Incubate on ice for 5 min.
  7. Centrifuge for 5 min at 500 x g.
  8. Save the supernatant. This is the Cytoplasmic Fraction.
  9. Resuspend pellet in 500 μl of MIB.
  10. Vortex for 15 sec.
  11. Incubate on ice for 5 min.
  12. Centrifuge for 5 min at 8,000 x g.
  13. Save the supernatant. This is the Membrane and Organelle Fraction.
  14. Resuspend pellet in 250 μl of CyNIB.
  15. Sonicate for 5 sec at 20% power 3 times. This is the Cytoskeletal and Nuclear Fraction.

F. Western Blot

  1. Add 60 μl of 3X SDS Loading Buffer with DTT (#7722) for every 100 μl of supernatant.
  2. Boil each sample for 5 min at 95°C and centrifuge for 3 min at 15,000 x g.
  3. Load 15 µl of each fraction along with 15 µl of WCL.

Table 1: Volumes in μl for WCL or buffer at indicated cell numbers.

Cell Count
2.5 x 106 cells 5 x 106 cells 7.5 x 106 cells 1 x 107 cells
WCL 50 100 150 200
CIB 250 500 750 1000
MIB 250 500 750 1000
CyNIB 125 250 375 500
Product Includes Quantity
Cytoplasmic Isolation Buffer (CIB) 10 ml
Membrane Isolation Buffer (MIB) 10 ml
Cytoskeletal/Nuclear Isolation Buffer (CyNIB) 5 ml
Protease Inhibitor Cocktail (100X) 5871 250 µl

Product Description

The Cell Fractionation Kit is designed to provide a fast and efficient way of separating cultured cells into three distinct fractions: cytoplasmic, membrane/organelle, and nuclear/cytoskeletal. These fractions can then be analyzed by SDS-PAGE and western blotting. The kit includes enough buffer for 20 assays.

Product Usage Information

Storage: Store at -20°C. Upon receipt, Protease Inhibitor Cocktail 5871 should be stored at 4°C. All other components should be stored at -20°C.

Cellular fractionation allows for the extraction of cellular proteins into distinct compartments. This is achieved by the use of detergents that take advantage of the inherent qualities and composition of different cellular membranes (1). Cellular fractionation has been important for defining the localization of many proteins, observing the translocation of proteins, and determining protein-protein complexes, such as cytoskeletal-associated proteins (2,3). Thus, detergent-based cellular fractionation separates cellular components with greater ease and speed compared to a more laborious density centrifugation method (4).

1.  Lenstra, J.A. and Bloemendal, H. (1983) Eur J Biochem 135, 413-23.

2.  Banno, A. et al. (2012) J Biol Chem 287, 13799-812.

3.  Loo, L.H. et al. (2009) J Cell Biol 187, 375-84.

4.  Michelsen, U. and von Hagen, J. (2009) Methods Enzymol 463, 305-28.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Cell Fractionation Kit