Technical

How to choose loading control antibodies
source:Technical divisiondate:2022-05-19views:932

 

The loading control antibody can be used to evaluate the efficiency of Western Blot and compare the amount of protein loaded per well in the gel. Such controls can help identify differences in expression levels between samples, whether due to actual protein levels in cell lysates or differences in loading.

Key functions of loading control antibodies

(1) Check if the whole WB process is in order

(2) Testing for correct gene expression products

(3) Comparison of relative changes in trace amounts of products

(4) Evaluate whether the total amount of protein in each well is generally consistent

Figure 1 Example

Loading control antibodies are also used as positive controls for co-staining in immunofluorescence studies of target proteins. Loading control antibodies are divided into unlabelled and labelled types, and markers include biotin, HRP, FITC, PE, Cy dyes and Alexa Fluor dyes.

There are many types of loading control antibodies, such as β-actin, β-tubunlin, GAPDH, Vinculin, LaminB, PCNA, etc. The selection of different loading control antibodies for different samples is also the key to the success of WB experiments. This article will briefly describe five principles that should be followed in the selection of loading control antibodies.

 

I. Source of sample species.

The first thing to consider is what species the experimental sample comes from.

 

1. Mammalian tissue or cell samples, usually β-actin, β-tubulin, GAPDH, Lamin B, Histone H3, Na,Katpase etc. are selected.

 

2. For experimental samples of plant origin, plantactin, Rubisco, etc. are available.

 

3. There are fewer studies of samples from other sources, so it is appropriate to refer to journal literature data and select appropriate proteins as loading controls.

 

II. Molecular weight size.

When selecting a loading control antibody, the molecular weight of the target protein should be taken into account, generally ensuring that the difference between the molecular weight of the target protein and the loading control protein is more than 5 KD. For example, if the molecular weight of the target protein is 40 KD, it is not appropriate to choose β-actin or GAPDH as the loading control, but rather α-Tubulin or β-tubulin.

 

III. Level of expression.

The loading control protein selected needs to be highly expressed in the sample to be tested. Commonly used loading control proteins are those that are highly expressed by housekeeping genes and are required for cell development and maintenance of cell viability. For example, PCNA, an loading control protein in the nucleus, is expressed in the S phase of DNA and is therefore not recommended for non-proliferating cells.

 

IV. Target protein expression localization.

 

 

 

V. ELK Biotechnology Optional Related loading controls.

Scope of application

Name

Item No.

Molecular weight

cytoplasm

β-Tubulin

EM1114

50kDa

β-Actin

EM1031

43kDa

Alpha-Tubulin

EM1115

52kDa

GAPDH

EM1028

37kDa

Vinculin

ES3700

123kDa

Cyclophilin B

EM1219

21kDa

Nuclear membrane proteins

Lamin B

EM1035

68kDa

Nucleoproteins

PCNA

EM1030

29kDa

TATA binding protein TBP/TBP

EM1216

38kDa

YY1

ES11233

45kDa

HDAC1

ES4225

55kDa

Histone H3

EM1037

15kDa

Membrane proteins

ATPase Na+/ K+ beta 2 (ATPB2)

/

112kDa

Plasma membrane

ATP1A1 (Sodium Potassium ATPase)

ES2892

100kDa

Mitochondria

COX4

EM1032

15kDa

VDAC1

ES4120

31kDa

Full blood/

Plasma/

Serum

Transferrin

EM1231

77kDa

Albumin

EM1190

66kDa

Plant

Plant actin

EM1147

43kDa