CALB1 antibody

Catalog No. ABIN1679483

The Rabbit Monoclonal anti-CALB1 antibody has been validated for WB and IF and has been independently validated. It is suitable to detect CALB1 in samples from Human.

Quick Overview for CALB1 antibody (ABIN1679483)

Target: CALB1 (Calbindin (CALB1))

Reactivity: Human

Host: Rabbit

Clonality: Monoclonal

Conjugate: This CALB1 antibody is un-conjugated

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

Product Details anti-CALB1 Antibody

Cross-Reactivity: Mouse, Rat

Characteristics: Monoclonal Antibodies

Purification: Affinity purification

Immunogen: A synthesized peptide derived from human Calbindin

Isotype: IgG

Application Details

Application Notes: WB,1:500 - 1:2000,IF,1:50 - 1:200

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104495 (Immunohistochemistry)

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

No.: #104495

Date: 08/02/2023

Antigen: CALB1

Lot Number: 4000000953

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 ABIN3018777 works in IHC-P on cultured cerebellar slices at 1:50 dilution.

Validation Images

Full Methods

Primary Antibody: ABIN3018777

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, ABIN3018777, lot 4000000953) diluted 1:50, 1:100, and 1:200 in 0.1 M PBS-BSA (Sigma, A7906)-PLL (Sigma, P1524) 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 (Invitrogen by Thermo Fisher Scientific, A11034, lot 2380031) diluted 1:500 in 0.1 M PBS for 1 h at RT.
  • 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, ABIN3018777, lot 4000000953) 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 #104531 (Western Blotting)

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

No.: #104531

Date: 08/02/2023

Antigen: CALB1

Lot Number: 4000000953

Method validated: Western Blotting

Positive Control:

Adult mouse cerebellum and microdissected Purkinje neurons

Notes:

Passed. The CALB1 antibody ABIN3018777 works in WB at a 1:1000 dilution with sensitive ECL substrate in adult mouse brain.

Validation Images

Western blot using ABIN3018777 to reveal CALB1 (28 kDa) in mouse laser-microdissected Purkinje neurons (1), and whole cerebellum (2).

Full Methods

Primary Antibody: ABIN3018777

Secondary Antibody:

goat anti-rabbit IgG (H+L) HRP-conjugated (Thermo Fisher Scientific, G-21234)

Full Protocol:

• Homogenize tissues and neurons 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 50 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, ABIN3018777, lot 4000000953) 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-rabbit IgG (Thermo Fisher Scientific, G-21234) 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.

Experimental Notes:

Non-specific bands were also detected in adult mouse cerebellum tissue extracts, but not on laser-microdissected Purkinje neurons.

Handling

Buffer: PBS with 0.02 % sodium azide,0.05 % BSA,50 % glycerol, pH 7.3.

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

Storage Comment: Store at -20°C. Avoid freeze / thaw cycles.

Target Details for CALB1

Target: CALB1 (Calbindin (CALB1))

Alternative Name: CALB1

Background: The protein encoded by this gene is a member of the calcium-binding protein superfamily that includes calmodulin and troponin C. Originally described as a 27 kDa protein, it is now known to be a 28 kDa protein. It contains four active calcium-binding domains, and has two modified domains that are thought to have lost their calcium binding capability. This protein is thought to buffer entry of calcium upon stimulation of glutamate receptors. Depletion of this protein was noted in patients with Huntington disease. [provided by RefSeq, Jan 2015],CALB, D-28K,Calcium Signaling,Cell Type Marker,Cell Type Marker_Neuron marker,Neuroscience,CALB1

Molecular Weight: 30 kDa

Gene ID: 793

UniProt: P05937