CALB1 antibody

Catalog No. ABIN6254097

Product Details anti-CALB1 Antibody

Target: CALB1 (Calbindin (CALB1))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This CALB1 antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)

Cross-Reactivity: Cow (Bovine), Human, Mouse (Murine), Rat (Rattus)

Purification: Protein G purified culture supernatant

Immunogen: Recombinant full length human calbindin purified from E. coli.

Isotype: IgG2a

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104494 (Immunohistochemistry)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #104494

Date: 08/02/2023

Antigen: CALB1

Lot Number: GS116G

Method validated: Immunohistochemistry

Positive Control:

Adult (>2 months) CD1 mouse cerebellum (6 µm glass-mounted microtome sections)

Postnatal day 6-7 CD1 mouse cerebellum (cultured cerebellar slices)

Negative Control:

Control slices were processed for each experimental procedure, omitting the primary antibody; overnight incubation in diluent solution only.

Notes:

Passed. The CALB1 antibody ABIN6254097 works in IHC-P at a 1:50 dilution with or without tyramide amplification.

Validation Images

Full Methods

Primary Antibody: ABIN6254097

Secondary Antibody:

goat anti-mouse IgG (H+L) AF488-conjugated antibody (Thermo Fisher Scientific, A11034, lot 2380031)

Full Protocol:

• Indirect IMF on microtome sections
• • Perfuse adult (>2 months) CD1 mice with paraformaldehyde 4% in 0.1 M phosphate buffer pH 7.4 and post-fix in the same fixative for an additional 2 h at RT.
  • Wash, dehydrate, and embed samples in paraffin wax.
  • Wash several times with 0.01 M PBS.
  • Cut the cerebellum with a microtome into 6 µm sections and mount them on glass slides.
  • After paraffin removal, incubate sections for 1 h at RT in PBS containing 1% albumin from chicken egg white (Sigma, A5378) and 0.3% Triton-X-100 (BioRad, 161-0407, lot 00583) to block non-specific binding sites.
  • Incubate sections with primary mouse anti-CALB1 antibody (antibodies online, ABIN6254097, lot GS116G) diluted 1:50, 1:100, and 1:200 in 0.1 M PBS-BSA-PLL ON at RT.
  • Wash 3x 5 min in 0.01 M PBS.
  • Incubate sections with secondary goat anti-mouse IgG (H+L) AF488-conjugated antibody (Fisher Scientific, A11034, lot 2380031) diluted 1:500 in 0.1 M PBS for 1 h at RT.
  • or alternatively with tyramide amplification:
  • Incubate sections with Poly-HRP-conjugated secondary antibody for 1 h at RT.
  • Wash sections 3x 5 min in 0.01 M PBS.
  • Incubate sections with Tyramide working solution (for 5 sections: 5 μL 100X Tyramide stock solution, 5 μL 100X H₂O₂ solution, 500 µL 1X Reaction buffer) for 10 min at RT.
  • Stop the reaction with the Reaction Stop Reagent working solution.
  • Wash 3x 5 min in 0.01M PBS.
  • Mount specimens in Fluoroshield (Sigma-Aldrich, F6182, lot MKCB0153V).
  • Acquire Images with Leica DM 6000B fluorescence microscope equipped with a digital camera at 20-40x magnification.
  
  
• Indirect IMF on cultured cerebellar slices
• • Euthanize CD1 mice at postnatal day 6-7 (P6-P7) with an overdose of 60 mg⁄100 g body weight sodium pentobarbital (Merck Life Science, Y0002194).
  • Remove the brain removed from the skull while the head is kept submerged in ice-cooled Gey’s solution (Sigma-Aldrich) supplemented with glucose and antioxidants (for 500 mL: 4.8 mL 50% glucose, 0.05 g ascorbic acid, 0.1 g sodium pyruvate).
  • Dissect the cerebellum from the brain.
  • Cut 350 μm thick parasagittal slices of the cerebellum with a McIlwain tissue chopper (Brinkmann Instruments).
  • Plate two to three slices onto a Millicell Cell Culture Insert (Merck Life Science, PICM0RG50).
  • Place each insert inside a 35 mm Petri dish containing 1 mL of culture medium consisting of 50 % Eagle basal medium (BME, Sigma Chemicals), 25 % horse serum (Gibco by Thermo Fisher Scientific), 25 % Hanks balanced salt solution (Sigma-Aldrich), 0.5 % glucose, 0.5 % 200 mM L-glutamine, and 1ؘ % antibiotic/antimycotic solution.
  • Incubate slices at 34 °C in 5 % CO₂ for up to 20 d in vitro (DIV). Change the medium twice a week. Slices were allowed to equilibrate to the in vitro conditions for at least 4-6 DIV before IMF.
  • Remove the culture medium from the dish and fix the slices in 1 mL of 4 % paraformaldehyde (Merck Life Science, P6148) in PBS for 1 h.
  • Wash 3x 5 min in 0.01 M PBS.
  • Incubate fixed cultures in PBS containing 1 % Triton X-100 (BioRad, 161-0407, lot 00583) for 10 min.
  • Wash 3x 5 min in 0.01 M PBS.
  • Incubate cultures ON at 4 °C under continuous stirring in PBS containing 1 % albumin from chicken egg white (Sigma, A5378) and 0.3 % Triton-X-100 (BioRad, 161-0407, lot 00583) to block non-specific binding sites.
  • Incubate cultures with the primary mouse anti-CALB1 antibody (antibodies online, ABIN6254097, lot GS116G) diluted 1:50 in PBS-BSA (Sigma, A7906)-PLL (Sigma, P1524) ON at RT.
  • Wash 5 x 5min in PBS.
  • Incubate cultures with the secondary anti-rabbit antibody Alexa Fluor 488 diluted (Invitrogen by Thermo Fisher Scientific, A11034, lot 2380031) 1:500 in 0.1 M PBS for 1 h at RT.
  • Wash 3x 5 min in 0.01 M PBS.
  • Mount specimens in Fluoroshield (Sigma-Aldrich, F6182, lot MKCB0153V).
  • Acquire Images with Leica DM 6000B fluorescence microscope equipped with a digital camera at 20-40x magnification.

Experimental Notes:

For indirect IMF on cerebellum paraffin sections, antigen retrieval treatment was also tested. In this case, sections were processed for microwave antigen retrieval for 10 min (95-100 °C) in 10 mM sodium citrate buffer (pH 6.0). After 20 min of spontaneous cooling, sections were washed twice for 5 min with distilled water and twice for 5 min with PBS.

Independent Validation (WB)

Validation #104530 (Western Blotting)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #104530

Date: 08/02/2023

Antigen: CALB1

Lot Number: GS116G

Method validated: Western Blotting

Positive Control:

Adult mouse brain and cerebellum

Notes:

Passed. The CALB1 antibody ABIN6254097 works in WB at a 1:1000 dilution.

Validation Images

Western blot results using ABIN6254097 to reveal CALB1 (28 kDa) in the adult mouse brain and cerebellum.

Full Methods

Primary Antibody: ABIN6254097

Secondary Antibody:

goat anti-mouse IgG (H+L) HRP-conjugated (Thermo Fisher Scientific, G-21040)

Full Protocol:

• Homogenize tissues with cold lysis buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1% protease inhibitor (Sigma P8340) using an ultrasonic homogenizer (MSE, SoniPrep 150) with 16 amplitude, 20 s on, 10 s off pulse for 60 s.
• Centrifuge tissue homogenates at 13,000 rpm for 20 min at 4 °C.
• Collect supernatants and determine total protein content using a Bradford assay.
• Denature 100 µg of total protein for 5 min at 90 °C and subsequently separate them on a denaturing 12% PAGE-SDS gel alongside a Precision Plus Protein Dual Color Standard (Bio-Rad, 160374).
• Electro-transfer proteins onto nitrocellulose membrane (Amersham Biosciences, RPN203D) ON in the cold room.
• Wash membrane 3x for 10 min with 0.01 M PBS containing 0.1% Tween-20 (PBST).
• Block membrane with PBST containing 2% bovine serum albumin for 1 h at RT.
• Incubate membrane with primary rabbit anti-CALB1 antibody (antibodies-online, ABIN6254097, lot GS116G) diluted 1:1,000 in PBST ON at 4 °C.
• Wash membrane 3x 10 min with PBST.
• Incubate membrane with secondary HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, G-21040) diluted 1:50,000 in PBST for 1 h at RT.
• Wash membrane 3x 10 min with PBST.
• Visualize proteins with SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, A38555) using a ChemiDoc Imaging System.

Handling

Buffer: 100 μL in PBS + 50 % glycerol and 5 mM Sodium azide

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Target Details for CALB1

Target: CALB1 (Calbindin (CALB1))

Alternative Name: CALB1 (CALB1 Products)

Synonyms: CALB antibody, CG6702 antibody, Calb antibody, Cbp33 antibody, DCBP antibody, Dcbp antibody, Dcp antibody, Dmel\\CG6702 antibody, cbn antibody, cbp53E antibody, Calbindin antibody, CB antibody, Calb-1 antibody, Brain-2 antibody, CaBP28K antibody, calb1 antibody, CALB1 antibody, fj53a05 antibody, wu:fj53a05 antibody, calbindin 1 antibody, Calbindin 53E antibody, CALB1 antibody, Cbp53E antibody, Calb1 antibody, calb1 antibody

Molecular Weight: '28 kDa

Gene ID: 793

UniProt: P05937