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60059
Complement C4-A/C4-B (E6J7W) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Complement C4-A/C4-B (E6J7W) Rabbit mAb #60059

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  1. WB
Western Blotting Image 1: Complement C4-A/C4-B (E6J7W) Rabbit mAb
Western blot analysis of extracts from various cell lines and tissues using Complement C4-A/C4-B (E6J7W) Rabbit mAb (upper) and β-Tubulin (D2N5G) Rabbit mAb #15115 (lower).
To Purchase # 60059S
製品番号 サイズ 価格 在庫
60059S
100 µl

Supporting Data

REACTIVITY H R
SENSITIVITY Endogenous
MW (kDa) 210
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

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
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 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.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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

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. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. 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: 263

Specificity / Sensitivity

Complement C4-A/C4-B (E6J7W) Rabbit mAb recognizes endogenous levels of total C4-A/C4-B protein. The predicted epitope of Complement C4-A/C4-B (E6J7W) Rabbit mAb is the N-terminus of the β-subunit of the C4-A/C4-B protein.

Species Reactivity:

Human, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro148 of human C4-A/C4-B protein.

Background

Complement is a collection of soluble proteins secreted in the blood and other body fluids (1). As part of the innate immune system, complement proteins are involved in the clearance of pathogens and damaged cells in a process called opsonization, which results in the coating of a pathogen with antibodies and/or complement proteins to facilitate phagocytosis of debris or foreign pathogens.  Complements are activated by a cascade of cleavage reactions, triggered initially by pattern recognition receptor-mediated detection of pathogens/debris. The cascade of cleavage-mediated products activate three distinct effector pathways, including inflammation, phagocytosis, and membrane attack that represent a coordinated defense of the host organism. Several complement proteins are encoded in the mammalian genome, designated by the capital letter C followed by a number, in order by their discovery. Complement C4-A and C4-B, encoded by the C4A and C4B genes, respectively, are the non-enzymatic components of C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. The C4A/C4B genes generate C4-A/C4-B polypeptide proteins, composed of an alpha, beta, and gamma-chain subunits linked by disulfide bonds. The polypeptide protein can be further cleaved to generate a cascade of fragments to initiate the innate immune response.  In addition to the innate immune response, several components of the complement system, including C4-A, have been implicated in brain development, as well as neurodevelopmental and neurodegenerative diseases. The complement system plays a role in microglial-dependent synapse pruning of excess synapses during development, particularly in the visual system (2). Additionally, complement-mediated synaptic pruning may also contribute to psychiatric diseases such as schizophrenia (3,4).
  1. Ricklin, D. and Lambris, J.D. (2016) Immunol Rev 274, 5-8.
  2. Stephan, A.H. et al. (2012) Annu Rev Neurosci 35, 369-89.
  3. Sekar, A. et al. (2016) Nature 530, 177-83.
  4. Ruzzo, E.K. and Geschwind, D.H. (2016) Nat Neurosci 19, 523-5.

Pathways & Proteins

Explore pathways + proteins related to this product.

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