I need a suitable loading control antibody that I can use in western blot of rat nerve extracts, which do you recommend?

This GAPDH antibody (NB300-221) is a good choice for a loading control antibody and has been cited in over 180 publications and used on rat lysates with good results.

I'm looking for a loading control between 24kDa - 65kDa for use in simple western, we tried an actin antibody we already had but it didn't work. What can you recommend?

Our GAPDH antibody NB300-221 has been validated in simple western and detects around 35 kDa so I think it would be a good choice for a simple western loading control antibody based on your size requirements.

I want to know why GAPDH is used as a loading control antibody in WB techniques.

Our GAPDH antibody makes a good loading control because GAPDH is a housekeeping gene essential to metabolism that is globally expressed in most tissue types and cell lines.

What secondary antibody should I use for visualization of the GAPDH antibody?

This GAPDH antibody is raised in mouse so you would want to use an anti-mouse secondary with the dye of your choice.

We received GAPDH antibody NB600-502 and there was only about 15µl of antibody in the tube. I was under the impression this should have been 100µl.

The unit size of this product is 0.1 mg with a concentration of 6.7 mg/ml. Hence the volume should be no less than 14.9 ul.

I wanted to know which of the two housekeeping genes B-actin or GAPDH can be used for best results. I intend to do an experiment to determine expression of IL-6 and TNF-alpha in liver and kidney tissues.

For homogenized tissue, beta-actin and GAPDH are both fine.

We need a loading control antibody for WB working with HeLa cell line, will this GAPDH antibody work for that?

GAPDH is expressed in HeLa cell lines but whether or not it is the best option for a loading control antibody depends on the size of the protein you are interested in. GAPDH antibody is a good choice for low to mid MW targets, as GAPDH detects around 35 kDa.

Do you offer a smaller size for the GAPDH antibodies?

We offer sample sizes for many of our GAPDH loading control antibodies including NB300-221.

Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

Name: Human/Mouse/Rat Total GAPDH DuoSet IC ELISA
Brand: R&D Systems
SKU: DYC5718-2

R&D Systems, part of Bio-Techne | Catalog # DYC5718-2

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Product Summary
Product Specifications
Scientific Data
Kit Contents
Other Reagents Required
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

156-10000 pg/mL

Sample Type

Cell Lysates

Reactivity

Human, Mouse, Rat

Human/Mouse/Rat Total GAPDH DuoSet IC ELISA Features

Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
Development protocols are provided to guide further assay optimization
Assay can be customized to your specific needs
Available in 2, 5, and 15- (96-well) plate pack sizes
Economical alternative to Western blot

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Product Summary for Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure GAPDH in cell lysates. An immobilized capture antibody specific for GAPDH binds both phosphorylated and unphosphorylated GAPDH. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

Cell lysates (100 µL)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

Standard Curve

Kit Contents for Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

Capture Antibody
Conjugated Detection Antibody
Calibrated Immunoassay Standard or Control
Streptavidin-HRP

Other Reagents Required

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂O₄, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Lysis Buffer*

IC Diluent*

Blocking Buffer*

Substrate Solution: 1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H₂SO₄ (Catalog # DY994)

Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent

*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: GAPDH

GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase) is a relatively stable enzyme involved in the second phase of glycolysis. It is generally thought to be present at a constant level in cells, regardless (in most cases) of experimental treatment or technical procedure. For this reason, measurement of GAPDH is generally used as an internal control for experimental error.

Long Name

Glyceraldehyde-3-phosphate Dehydrogenase

Alternate Names

G3PDH

Entrez Gene IDs

2597 (Human); 14433 (Mouse); 24383 (Rat)

Gene Symbol

GAPDH

Additional GAPDH Products

Product Documents for Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

Product Datasheets

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COA

SDS

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Product Specific Notices for Human/Mouse/Rat Total GAPDH DuoSet IC ELISA

For research use only

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