Ho much protein did you use? We usually use about 30µg. 

And did you use a protein ladder? If yes, I would suggest the problem within the gel, as you should be able to see the ladder.

Your gel looks like there was hardly any protein in the samples and the small amount of protein you had seemed to stay in the well. How did you prepare your 4% mechanically deboned chicken meat protein solution and how did you treat it for loading onto the SDS PAGE? Also, a very high salt concentration, lack of reducing agent and low levels of denaturation can potentially lead to smeary bands and proteins that aggregate in the well without running into the gel ... but you should still be able to see some bands, which makes me think the problem lies in your sample rather than the gel.

Hey...

I would say you have low protein concentration, judging by the looks of the wells, but the protein that is being loaded should be enough. So, I believe  the problem is that it is deeply aggregated/it has an enormous native molecular weight. 

I think the solution is to use a more intense denaturation:

-> protein + loading buffer

->5min at 100ºC

->let it cool down, homogenize well, load the sample in the gel

Also, the loading buffer should contain Urea, at a concentration between 2M and 8M. 

Good luck!

Dear Dr. Ariza,

I am extracting proteins with alkali solubilization and acid precipitation procedure from mechanically deboned chicken meat. For this purpose, I am mixing meat with distilled water at a ratio of 1:9. I am adjusting pH to 12 with 5 M NaOH, which is the best soluable pH, and mixing it with magnetic stirrer for 1 hour. Then, solution is centrifuged at 9000 rpm for 30 min, and the pH of supernatant is adjusted to isoelectric point of meat proteins. Precipitated proteins are centrifuged at 9000 rpm for 10 min, and then dried at 40 oC in an air circulated oven. Protein concentrate consists of 80% protein, fat content is minimazing with soxhlet extraction.

For preperation of 4% protein solution, 5 g protein concentrate is mixing with 100 mL distilled water, and pH of slurry is adjusting to 12. This is the preparation procedure of 4% mechanically deboned chicken meat protein.

Dear Dr. Ziegler,

I loaded bovine serum albumin and saw protein bands. So, there is no problem for gels.

Please help for solving this problem.  

It is almost imposible to clearly see what happened. There are two options: the simple or the technique. It is recomended to run protein standards, so we can decide whether it is the simple or the technique. It is posible that the amount of proteins in your simple be quite low. have you quantified them? It is not a matter of volumen added but the amount of proteins. Even so, you get a band that did not migrate at all. Check the acrylame concentration in the resolving gel. Finnaly, what is the expected gel image? Have a look to the literatura.

.

Are you sure there is protein in your sample? Perform a suitable protein quantitation assay to find out and, additionally, you will be able to load a specific amount of protein (for example, 20 ug)

Dear Furkan, It seems like you should at least have some protein according to your method. As has been mentioned already, run a quantification assay (e.g. Bradford) on your samples to see if you still have soluble protein after resuspending your protein concentrate. You should always run a protein ladder with your sample when you run an SDS PAGE gel ... otherwise, how will you know the approximate sizes of your different protein bands? If you buy a commercial ladder and don't see it on the gel, then you have a problem with the gel. If you see the ladder but you don't see your samples, then there is a problem with your sample preparation.

You seem to simply resuspend the dried protein in distilled water and nothing else ... maybe your protein sample doesn't like that and therefore doesn't go into solution, which is why it seem to stay in the well. Usually "whole" protein solutions (solutions containing a mixture of different proteins, all extracted at the same time from the same sample) are prepared in buffers with a slightly less alkaline pH than 12 (e.g. TRIS, HEPES, or phosphate buffer with pH 7 to 9) that will suit most of the proteins in an average tissue sample. The buffer should also contain some ionic compound (e.g. 150 mM NaCl) as most proteins have charged surfaces and some of them can precipitate in a non-ionic environment. You should also add a reducing agent (e.g. 2 mM DTT) to lower the probability of aggregation via disulphide bonds. The loading dye for SDS PAGE gel should help solubilise your protein even further, as it should be denatured by the SDS in the dye and by heating (3 to 5 minutes at 97 to 100 degrees C after mixing with the loading dye) prior to loading the sample.

Run with standard proteins and determine the amount of proteins in your samples before electrophoresis

It looks like you did not have enugh protein or that the proteins were aggregated in the well. You have to check your sample preparation and protein concentrations before you load. Also include a standard to which you can compare to.

I doubt this has anything to do with your samples. Your gel is obviously cloudy, even in the well separators, so there is no way you can assess your samples. If you ran MW markers, why did they not run correctly? The gel is bad. Run another one. If it is not crystal clear everywhere except the sample lanes, something is wrong with your gel preparation solutions, and you do need to start over with new reagents.

Hi! I agree with the others. I think the sample is too diluted. Check for protein concentration using Bradford or BCA assay. I had the same problem when I mechanically prepared my rat brain sample in too much buffer. Maybe you can reduce the volume of the lysis buffer or find an alternative lysis buffer which works better. I used to use RIPA. But maybe won't work on the chicken meat protein. Good luck!

According to your description, your sample pH is around 12. This might be the reason.I´m pretty sure your sample buffer will not bring the pH down to 6.8 and thus you may get anomalous migration. Also, there might be to much salt in your sample. Try to remove your salt and lower the pH by dialysis. You can also, just for analytical purposes, precipitate your samples with trichloroacetic acid See the complete protocol at : http://www.its.caltech.edu/~bjorker/TCA_ppt_protocol.pdf

Hope that helps 

http://www.its.caltech.edu/~bjorker/TCA_ppt_protocol.pdf