How to select a proper loading control antibody for Western Blot?
Loading control is also called internal control. In terms of mammalian cell expression, it generally refers to the protein encoded by housekeeping genes (Housekeeping Proteins). Their expression in various tissues and cells is relatively stable. It is often used as a reference while testing the expression level of a specific protein. In the Western Blotting experiment, it is proposed to do a internal control detection which can correct protein quantification and make up for the experiment error in loading samples procedure, ensuing the accuracy of the experimental results.
There are two kinds of method about how to use loading control antibody in Western Blot.
1.Conjugated loading control antibody detection: Add HRP conjugated loading control antibody in secondary antibody incubation.
2.Normal loading control antibody detection:
- If molecular weight(MW) is similar between your targeted protein and selected loading control protein, it is proposed to do the detection of target protein firstly, then using strip buffer to wash the antibody in membrane, and doing the detection of loading control antibody.
- If molecular weight(MW) difference is obvious between your targeted protein and selected loading control protein, using prestained protein marker after transmembrane, according to the size of protein marker, then cut off the membrane into two parts, high molecular weight and low molecular weight, dividing your target protein and loading control protein. Finally do Incubation in separate membrane for your primary antibody and loading control antibody, secondary antibody incubation and color development.
There are many kinds of loading control, so choose a right loading control for your experiment is very important. Below points can help you to better make the decision.
- Sample species: For tissues and cells of mammals, it is generally recommended to choose the proteins like beta-actin, Tubulin, GAPDH, Lamin B, PCNA, Na+/K+-ATPase, etc. Plant actin and Rubisco are often used in plant samples.
- Molecular weight of the target protein: Select a loading control that has a different molecular weight with the protein of interest (>5 kDa), so that you could distinguish between the bands. For high molecular weight (>100 kDa) proteins, we recommend Vinculin as a loading control.
- Expression location of the target protein: whole cell, nuclear, mitochondrial, membrane, etc..
- In some certain conditions, the expression of loading controls will be abnormal in some cells, this should be taken into account.
Abbkine offers a wide range of loading control antibodies, including the hot targets and unique species, covering the molecular weights from 15 kDa to 116 kDa. Also our conjugated loading control antibodies have diverse conjugates, including enzyme and featured fluorescent dyes, with the application of WB, IHC, IF and IP experiments.
Taking a specific example here, if you extract total proteins or cytoplasmic proteins from cells, beta-actin, GAPDH and Tubulin will be your choice, and luckily Abbkine can provide a series of antibodies helpful for your research, kindly refer to below:
|A01010||Anti-β-Actin Mouse Mab (1C7)||43 kDa||IF, IHC, WB
|A01020||Anti-GAPDH Mouse Mab (2B5)||37 kDa||IF, IHC, WB||47|
|A01030||Anti-β-Tubulin Mouse Mab (3G6)||55 kDa||IF, IHC, WB||8|
|A01080||Anti-α-Tubulin Mouse Mab (3G5)||55 kDa||WB, IHC, IF, IP|
|A01130||Anti-Cyclophilin B Mouse Mab (7B2)||24 kDa||WB, IHC, IF|
If you have any further interests to learn Abbkine loading control antibody category, kindly refer at https://www.abbkine.com/producttype/loading-control-antibody/, send an E-mail to email@example.com, our representative will surely contact you back shortly.
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