The introduction of antibodies that specific to internal control protein (I)

Western Blot is a kind of experiment which is usually used for primarily verifying gene encoded products and discriminating the difference of protein quantity among different samples. As Western Blot includes lots of steps and reagents, when the result is not consistent to the predicted one, especially, all negative result or all positive results were shown, it is a very tough task to analyze what is wrong in the experiment. Based on this, it is recommended that a suitable reference system should be set up in a strict Western Blot experiment, and the reference system will be helpful for result analysis. A common used reference system includes several references, for instance, molecular weight (MW) marker, plasmid control, negative and positive control sample, internal control and so on, the internal control is an essential reference factor.

Internal Control is a reference in the experiment system, regularly, internal control in mammalian cell expression system are proteins expressed by house keeping gene. These proteins show relatively constant quantity in different tissue and cells. So, when Western Blot is designed for protein level test, the internal control proteins could be considered as a reference to determine the consistence of samples, for instance, quantity of tissue, cell, loading sample, etc, and correct the operate error in the experiment, to further ensure the accuracy of the result. So, internal control data is very important for Western Blot.

Currently, the common used internal controls include beta-actin，beta-tubulin， GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and so on. In order to distinguish the target molecule and internal control, it is recommended that the difference between the MW of the two proteins should be larger than 5KD.

Actin is an important cytoskeletal protein, it includes 6 subtypes, including alpha-skeletal muscle actin, alpha-cardiac muscle actin, alpha-smooth muscle actin, gamma-smooth muscle actin, beta-actin (β-non-muscle) and gamma-non-muscle actin. The first four actin are tissue specific, but the later two ones are widely expressed in different tissue. The common used beta-actin, whose MW is 42kd, shows a very low abundance in muscle, so, if the immunogen using to prepare antibody is beta-actin specific peptide (for instance, N-terminal peptide), then the antibody might not detect positive signal in muscle tissue, while the immunogen is peptide possessing high homology with other actins, then the antibody will detect positive signals in tissue including muscle. Different from antibodies prepared by N-termnal and C-terminal peptides in abcam and sigma, Cloud-Clone Corp. prepared antibody specific to beta-actin (PAB340Mi) by using full-length protein. The result of Western Blot shows that the antibody can recognize beta-actin in different tissues including heart muscle, at the meanwhile, due to the high homology of this protein in different species, the antibody could be used for detection of internal controls in samples from different species.

Tubulin is another important cytoskeletal protein, there are lots of subtypes, including alpha, beta, gamma, delta and epsilon tubulin. The beta-tubulin whose MW is about 50kd shows many similar characteristics with beta-actin, so, it is a common used internal control in Western Blot, as well. Antibody specific to beta-tubulin (PAB870Mi01) prepared Cloud-Clone Corp. had been used by lots of researchers in Western Blot test.

GAPDH, a common used internal control protein, is an important enzyme in glycolysis with MW of about 36kd. Antibody of PAB932Hu01 and PAB932Hu02, whose immunogen are recombinant protein including amino acids of Gly2~Ser148 and Thr154~Val324, could be used as antibody for internal control protein, GAPDH. Please notice that the level of GAPDH will increase in tissue hypoxia and diabetes, so, GAPDH is not suitable to be used as internal control for these samples in Western Blot.

As for internal control for nucleus, Cloud-Clone Corp. had developed antibodies specific to Histone H3 and proliferating cell nuclear antigen (PCNA). PAA285Mi01 is specific to chromosome constitutive expressed H3, and PAA591Mi01 is specific to PCNA which is synthesized in nucleus. They had been used as internal control antibodies in Western Blot for numerous researchers.

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