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63735
MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen)

MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen) #63735

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APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 80 Rabbit IgG
Western Blotting
Western Blotting

Western blot analysis of extracts from various cell lines using MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen) (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, HCT 116 cells are negative for MLL2 expression.

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IP
Immunoprecipitation

Immunoprecipitation of MLL2/KMT2B from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen). Western blot analysis was performed using MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen).

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IF-IC
Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of A549 cells (left, positive) and HCT 116 cells (right, negative) using MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

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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

Protocol Id: 10

PRINT

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing magnetic separation.

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.

  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X 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.
  4. Protein A Magnetic Beads: Use Protein A (#73778) for rabbit IgG immunoprecipitation.
  5. Magnetic Separation Rack: (#7017) or (#14654).
  6. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

  1. Briefly vortex the stock tube to resuspend the magnetic beads.

    IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:

  2. Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

    Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

  3. Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

    IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.

  4. Incubate with rotation for 20 minutes at room temperature.
  5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
  6. Proceed to immunoprecipitation section.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex.
  2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
  3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
  4. Incubate with rotation for 20 min at room temperature.
  5. Pellet beads using magnetic separation rack. Wash pellets five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  6. Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
  2. Heat the sample to 95-100°C for 5 min.
  3. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15-30 µl) on SDS-PAGE gel.

posted December 2008

revised October 2017

Protocol Id: 410

PRINT

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

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

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.

  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 24

Application Dilutions
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:1000
Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

MLL2/KMT2B (D6X2E) Rabbit mAb (Carboxy-terminal Antigen) recognizes endogenous levels of total MLL2 protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human MLL2/KMT2B protein. This antibody detects the full-length 600 kDa MLL2/KMT2B protein and the Taspase 1-cleaved 80 kDa C-terminal MLL2/KMT2B protein (MLL2/KMT2B-C).

The Set1 histone methyltransferase protein was first identified in yeast as part of the Set1/COMPASS histone methyltransferase complex, which methylates histone H3 at Lys4 and functions as a transcriptional co-activator (1). While yeast contain only one known Set1 protein, mammals contain six Set1-related proteins: SET1A, SET1B, MLL1, MLL2, MLL3, and MLL4, all of which assemble into COMPASS-like complexes and methylate histone H3 at Lys4 (2,3). These Set1-related proteins are each found in distinct protein complexes, all of which share the common subunits WDR5, RBBP5, ASH2L, CXXC1, and DPY30, which are required for proper complex assembly and modulation of histone methyltransferase activity (2-6). MLL1 and MLL2 complexes contain the additional protein subunit, menin (6).

MLL2, also known as histone-lysine N-methyltransferase 2B (KMT2B), functions to activate gene expression by mediating tri-methylation of histone H3 lysine 4 at the promoters of genes involved in embryogenesis and hematopoiesis, and is required for histone H3 lysine 4 tri-methylation at bivalent promoters in embryonic stem cells (7). Like MLL1, MLL2 is a large protein made up of approximately 2700 amino acids that is cleaved by the Taspase 1 threonine endopeptidase to form N-terminal (MLL2-N) and C-terminal (MLL2-C) fragments, both of which are subunits of the functional MLL2/COMPASS complex. MLL2-N, MLL2-C, WDR5, RBBP5, and ASH2L define the core catalytic component of the MLL2/COMPASS complex, which is recruited to target genes to regulate transcription. MLL1 gene translocations are often associated with various hematological malignancies and thought to be a driving component of these types of leukemia. MLL2 is required for memory formation, proper glucose homeostasis, and cardiac lineage differentiation of mouse embryonic stem cells (8-11). A recent study has shown that MLL2 is required for survival of MLL-AF9-transformed cells, implicating MLL2 as a potential modulator of MLL1-rearranged leukemias (12). Mutations in MLL2 cause complex early-onset dystonia, and overexpression of MLL2 is associated with gastrointestinal diffuse large B-cell lymphoma (13,14).

  1. Miller, T. et al. (2001) Proc Natl Acad Sci U S A 98, 12902-7.
  2. Shilatifard, A. (2008) Curr Opin Cell Biol 20, 341-8.
  3. Tenney, K. and Shilatifard, A. (2005) J Cell Biochem 95, 429-36.
  4. Lee, J.H. and Skalnik, D.G. (2005) J Biol Chem 280, 41725-31.
  5. Lee, J.H. et al. (2007) J Biol Chem 282, 13419-28.
  6. Hughes, C.M. et al. (2004) Mol Cell 13, 587-97.
  7. Denissov, S. et al. (2014) Development 141, 526-37.
  8. Kerimoglu, C. et al. (2017) Cell Rep 20, 538-548.
  9. Kerimoglu, C. et al. (2013) J Neurosci 33, 3452-64.
  10. Goldsworthy, M. et al. (2013) PLoS One 8, e61870.
  11. Wan, X. et al. (2014) Stem Cell Rev 10, 643-52.
  12. Chen, Y. et al. (2017) Cancer Cell 31, 755-770.e6.
  13. Meyer, E. et al. (2017) Nat Genet 49, 223-237.
  14. Ye, H. et al. (2015) Int J Clin Exp Pathol 8, 13043-50.
Entrez-Gene Id
9757
Swiss-Prot Acc.
Q9UMN6
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
Tween is a registered trademark of ICI Americas, Inc.

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