Assuming the transfer was even/good, and the protein is loaded evenly, then perhaps GAPDH isn't expressed evenly across groups? It seems like the proteins on the right are expressed less, not sure if the right side and left side of the blot are different groups?

Try another control such as beta-actin

I would highly recommend you to try total protein normalization (TPN)! There are several possible reasons for your bands being differently in their intensity, the worst one already mentioned by Can Kiessling: Varying expression levels of "house hold" genes. You can avoid the cumbersome search for a protein NOT affected by your conditions by TPN and have reliable, reproducible results.

Many suppliers offer now systems for TPN, a good start for reading is this paper here:

Article Cy5 total protein normalization in Western blot analysis

You can do a Ponceau staining of that blot to see the total protein staining. Post-transfer, if total protein looks even, then you are lead to the conclusion that GAPDH is changing expression. I also agree with Benjamin that total protein is a superior normalization method for protein expression and would highly suggested that you conduct some research on this method. Various commercial vendors such as Licor and Bio-Rad have kits that you can try.

Article Stain Free Total Protein Staining is a Superior Loading Cont...

Can Kiessling Here is my ponceu stained membrane. Please have a look!

Hello, as is it's hard to tell. Perhaps a little bit of wash will be better? I've attached an image of a ponceau i found on google search. In that blot, you can clearly see equal protein.

Amended on 14 October 2019

I must sadly say that notorious SDS-PAGE, IEF, and Western blotting are bad in the Protein Biochemistry (please see file; IEF for Hydrophobic protein). Therefore, we must have developed an HPLC-Surfactant-SEC method (please see file; SEC column 300A silica), which works better than the non-quantitative SDS-PAGE.

Mouse Glyceraldehyde-3-phosphate dehydrogenase/GAPDH/Peptidyl-cysteine S-nitrosylase GAPDH (Mr 38,000) may be easily analyzed by using the high-recovery RP-HPLC (please see file; Lysozyme by RP-HPLC). Further, we must have developed a PDMD (Protein-Direct-Microsequencing-Deciphering) method, which quantitatively works well than the non-quantitative Western Blot and 2D electrophoresis/2D-EP (please see files; HepG2 Fucoidan and JMBT Alopecia).

PDMD (Protein-Direct-Microsequencing-Deciphering) method is the strong method, which can definitively conclude the absence of protein in the biological specimens. On the other hand, ELISA and/or RIA and PCR usually give false positive or wrong results.

Further, I must sadly say that Glyceraldehyde-3-phosphate dehydrogenase/GAPDH, Actin, Insulin, and Insulin receptor are not present in Humans (my unpublished observation). Since GAPDH/GA3P-DH is present in aerobic bacterium of Agrobacterium tumefaciens (please see file; Dr. C-K Chern Agrobacterium), Humans seems to be different to Aerobic bacterium due to famous Species Differences.

GAPDH/Peptidyl-cysteine S-nitrosylase GAPDH may be present in Mouse, but absent in Humans. Therefore, notorious Apoptosis may be present in Mouse, but absent in Humans. Thus, the Fucoidan effect or the Mozuku effect is not due to prevention of Apotosis, but lowering the growth velocity of cancer tissue in the outbred SD rat (Please see file; Rat DEN Np-Fuco). Species differences among Humans, Rat, and Mouse seem to be unexpectedly great.

Humans has following Glycolytic enzymes; i.e.,

Glucose-6-phosphatase/G6Pase, L-Lactate dehydrogenase A-like 6A, Alcohol dehydrogenase 6, Fructose-bisphosphate aldolase C/Brain-type aldolase, Alpha-Ketoglutarate dehydrogenase/2-Oxoglutarate dehydrogenase, mitochondrial/OGDH/OGDC-E1/2-Oxoglutarate dehydrogenase complex component E1, ATP-dependent 6-phosphofructokinase, liver type/ATP-PFK/Phosphohexokinase, Fumarase/Fumarate hydratase, L-Lactate dehydrogenase A chain/LDH-M, Phosphoenolpyruvate carboxykinase/PEPCK-C, Transketolase/TK, 6-Phosphogluconate dehydrogenase, decarboxylating/PGDH, NAD-dependent malic enzyme, mitochondrial/Malic enzyme 2/NAD-ME, ADP-dependent glucokinase, Glutamic-alanine transaminase 1/ALT1/GPT 1, Alpha-Ketoglutarate dehydrogenase-like, mitochondrial/OGDC-E1-like, and Pyruvate kinase PKM/Pyruvate kinase muscle isozyme.

Pyruvate kinase PKM/Pyruvate kinase muscle isozyme is physiologically working as a homotetramer. Importance of multimer formation is reported recently (please see file; The Fascio effect).

Therefore, Humans seems to have a new Glycolytic pathway without using Glyceraldehyde-3-phosphate dehydrogenase/GAPDH; i.e.,

Glucose > G6P > F6P > Fructose-bisphosphate/F1,6BP > Dihydroxyacetone phosphate/DHAP > Pyruvate > Lactate

Other pathway G6P > DHAP, alpha-KGA/alpha-Ketoglutarate > Pyruvate (by using ALT) seems to be also present.

Furthermore, I have recently found that Humans has 2-kind of Carboxylase; i.e., Propionyl-CoA carboxylase beta chain, mitochondrial/PCCase subunit beta/PCC, and

Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial/MCCase subunit alpha/MCC, but does not have Acetyl-CoA carboxylase/ACC and Pyruvate carboxylase /PC as assessed by the PDMD method (please see file; 4 kind of carboxylases). This may also be due to Species Differences.

HCC tissue with PBC has Arachidonate 12-lipoxygenase, 12S-type/12S-LOX/ALOX12/Arachidonate 12-lipoxygenase/12-Lipoxygenase/12S-Lipoxygenase/12-LOX/Platelet-type 12-lipoxygenase/Arachidonate:oxygen 12-oxidoreductase/Delta12-Lipoxygenase/12Delta-Lipoxygenase/C-12 Lipoxygenase/Leukotriene A4 synthase/LTA4 synthase.

ALOX12/Platelet-type 12-lipoxygenase is distinguished from Leukocyte-type 12-lipoxygenase/Arachidonate 15-lipoxygenase/15-LOX/12/15-Lipoxygenase/12/15-LO/Arachidonate 12-lipoxygenase, leukocyte-type/L-12LO/Arachidonate omega-6 lipoxygenase. Uniprot says that "Leukocyte-type 12-lipoxygenase/15-LOX is found in mice, rats, cows, and pigs but not humans". We have now found that Platelet-type 12-lipoxygenase is surely present in Humans, but Leukocyte-type 12-lipoxygenase is definitely absent in Humans by using our reliable PDMD method (our unpublished observation).

By the way, I would like to express my opinion about the notorious "Housekeeping" genes or "House hold" genes. I have previously found that Enzyme has Wide-range of substrate specificity, although stringent rules are present (please see file; Enk serBIN). Therefore, I am recognizing that many peptide-hormone receptors have simultaneously peptidase activity (my unpublished estimation). Then, the notorious idea of "Housekeeping" genes or "House hold" genes only are indicating "Multiple-substrates-recognizing enzymes". Therefore, the finding by Drs. Charng-Kuang Chern, Iwao Kusaka and Sakuzo Fukui that "the Mutation in the Pyruvate carboxylase gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates" is surely the important conclusion onto Procaryote (please see file again; Dr C-K Chern Agrobacterium).

Kou Hayakawa Maybe I got you wrong but just to clarify: Humans pretty damn sure DO express Actin and GAPDH. Sorry, I probably just misunderstood you, but I couldnt let that stand ;)

There is usually a significant downregulation of GAPDH gene after treatment (GAPGH gene expression is affected by cell stress) which induces cell stress.

Even if you repeat the procedure, you'll still have the same result.

Alternatively, try other "house keeping" gene in your PCR to be able to exclude the unexpected effects of the exposure/treatment.

Another around it is to compare the Ct-value in the exposed group vs unexposed group by using the control gene.

read this:

Bustin, Stephen A., et al. "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clinical chemistry 55.4 (2009): 611-622.

The GAPDH been an enzyme in the Glycolytic pathway will certainly have its expression altered if any exposure effects the energy metabolism of the pathway. It is not a gene of choice for normalization qRT-PCR data in spermatozoa for studying the  effect of cryopreservation. 

Hi R. Trivedi ,

I use stain free blots (BIORAD) to do loading controls. I agree with Can Kiessling and Benjamin König .

I think your ponceau staining needs be washed to improve contrast.

After that, scan your image and quantify the lane with imageJ or other software. Statistically, the load of your different experimental groups need to be equal.

If the load is equal, use the total protein signal to normalize your data.

Best,

Jacó