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58232
COL6A1 (E9Q3P) Rabbit mAb
Primary Antibodies
Monoclonal Antibody
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COL6A1 (E9Q3P) Rabbit mAb #58232

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  1. WB
  2. IF
Western blot analysis of extracts from various cell lines using COL6A1 (E9Q3P) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Negative expression of COL6A1 protein in HCT-15 cells is consistent with the predicted expression pattern.
Confocal immunofluorescent analysis of fixed frozen mouse spleen labeled with COL6A1 (E9Q3P) Rabbit mAb (green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with CD45 (D3F8Q) Rabbit mAb (Alexa Fluor® 555 Conjugate) #19581 (red).
Confocal immunofluorescent analysis of fixed frozen mouse small intestine labeled with COL6A1 (E9Q3P) Rabbit mAb (left, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with CD45 (D3F8Q) Rabbit mAb (Alexa Fluor® 555 Conjugate) #19581 (right, red). Sections were mounted in ProLong Gold Antifade Reagent with DAPI #8961 (right, blue).
Confocal immunofluorescent analysis of fixed frozen mouse kidney labeled with COL6A1 (E9Q3P) Rabbit mAb (left, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with AQP4 (D1F8E) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #89851 (right, red). Sections were mounted in ProLong Gold Antifade Reagent with DAPI #8961 (right, blue).
Confocal immunofluorescent analysis of PC-3 cells (left, positive) and HCT-15 cells (right, negative) using COL6A1 (E9Q3P) Rabbit mAb (green), DyLight 554 Phalloidin #13054 (red), and DAPI #4083 (blue).
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To Purchase # 58232
Cat. # Size Qty. Price
58232S		 100 µl

Custom Formulation

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa) 130
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Frozen) 1:3200 - 1:6400
Immunofluorescence (Immunocytochemistry) 1:3200 - 1:12800

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.

Protocol

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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 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. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  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 (#59329, 10 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 webpage) 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 June 2020

Protocol Id: 10

Immunofluorescence (Frozen Tissues with Citrate Retrieval)

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 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. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  4. 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) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  5. 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.
  6. Recommended Fluorochrome-conjugated Anti-mouse secondary antibodies:
  7. NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
  8. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation and Antigen Retrieval

NOTE: Application of traditional antigen retrieval procedures to fixed-frozen tissues may result in excessive liberation of sections from the slide. It is highly recommended that users pre-treat glass slides with a reagent that promotes tissue adherence.(e.g., HistoGrip™) while reducing retrieval temperature to 70°C.

  1. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6-20 µm and place on positively charged slides.
  3. Bake on a slide warmer at 60°C for 1 hour (this helps sections adhere to slides).
  4. Submerse slides in 1X citrate unmasking solution, heat in a microwave, and maintain a temperature of ~70°C for 20 minutes.
  5. Cool slides on bench top for 30 min.
  6. Rinse sections briefly in 1X PBS.

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 protocol on product webpage 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 October 2017

Protocol Id: 1569

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 product webpage 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

Specificity / Sensitivity

COL6A1 (E9Q3P) Rabbit mAb recognizes endogenous levels of total COL6A1 protein. Non-specific cytoplasmic labeling of Purkinje cells in the cerebellum, acinar cells in pancreas, and tubules in testis may be observed by immunofluorescence.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile536 of human COL6A1 protein.

Background

Collagen type VI (ColVI) is a microfibrillar collagen found in the extracellular matrix (ECM) of muscles, bone, and connective tissues (1). ColVI consists of three main alpha chains: alpha 1, alpha 2, and alpha 3, which are encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. The three alpha chains form triple helix monomers and tetramers, which further assemble into a beaded microfilament network structure in the ECM (1,2). ColVI interacts with other ECM components, such as collagens, fibronectins, and perlecan, to support ECM mechanical sensing, anchoring the basement membrane to the surrounding ECM and inhibiting apoptosis and oxidative damage (3,4). Mutations in each of the ColVI genes (COL6A1, COL6A2, and COL6A3) result in defective ColVI assembly, causing Ullrich congenital muscular dystrophy (CMD) and Bethlem myopathy due to malformation of ECM structure (5,6). Knockout of COL6A1 in mice displays a mild myopathy and neurodegeneration associated with mitochondrial dysfunction, defective autophagy, and spontaneous apoptosis of muscle fibers (7,8). Increased COL6A1 in the ECM promotes tumor growth, metastasis, and therapeutic drug resistance (9-11).

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For Research Use Only. Not for Use in Diagnostic Procedures.
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