What method are you using for protein purification?

If you can share the details, it would be easier to find out the reason.

Hello Divya,

It looks to me that or you have a bad protein purification process or you use to much stringent conditions for your western blot analysis, affecting the protein integrity. I recommend you have a look on the WB resources following this link to figure out what might be your problem : http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-fr/applications/western-blotting/

Regards

Hi! Thanks for your reply, it was purified through his tag Fusion.

Could you share some details of your experiment. It may help to find some clue.

Please answer few questions it would be easier to find out the problem:

Is your Restriction digestion was showing your band of interest?

Which vector did you use?

What was the IPTG concentration and time for induction?

How are you purifying the protein (Manual or ÄKTA Protein Purification Systems)

Have you optimized the Imidazole concentration?

After getting your answers, we can solve the problem.

Thanks

If molecular marker bands look good and your desired bands not good then it may be two possibilities. Either due to increase sample volume or sample loading dye.

If sample volume is too high for a well and expressed protein concentration is also high then it will not fit in single line and expands.

You need to use fresh loading dye. After mixing with protein, boil the sample for 5-10 minutes, then centrifuge at 8000 rpm for 5 min and load on gel.

If your molecular marker along with desired proteins bands looks too ugly then you need to focus on gel preparation materials and its %age, pH of ingredients, running buffer, 30% acrylamide purity. If all these materials are new and pH is also ok then just increase your gel %age. If using 8% then try 12% and if you are using 12% then change to 15%.

Hope you will get good pretty bands.

Hi Divya,

What protein are you using? Because its integrity is being affected by the experiment. I would need to know more details about the experiment to know where the problem was. But my first advice is:

Make sure that the protein purification is being performed properly!

When analysing the bands at the end the experiment, check on the marker. If you get smeary band everywhere but in the marker region, the problem is in the purification process.

lf all the bands, including the dying marker, look smeary the problem occurs when performing SDS-PAGE electrophoresis. The most probable cause would be that the marker loading is not being added properly. But It's also a good idea to make sure that the gel setting up is correct and that the electrophoresis tank works as it should.

In addition, It's convenient to emphasise that proteins with lots of hydrophobic regions or non-protein substituents (i.e. multi-pass transmembrane proteins) can create smeary regions using His-tag Fusion.

Hope you get good bands.

Isabel

the method of protein purification are the ones that determine that

Hi Divya,

It might be the issue with your electrophoresis protocol can you please share the image? It would be more easy if you share experimental details.

Smearing may be the reason of some problem with electrophoresis if this is the case then marker bands must have been normally resolved

if not then try to optimized your SDS-PAGE.

As you said there was no induced protein band visible in your gel, it seems that you have to first focus on optimization of induction step.Make sure that are using appropriate strain for expression also make sure the cloning occurred appropriately.

If all of the above are accurate then try to confirm if there would be no nucleic acid or bacterial cell wall contamination in lysate. Try to centrifuge or pass the lysate through sterilized 23 or 22 gauge syringe needle thrice, or centrifuge to separate cell debris also make sure the proper denaturation before loading.

Hope this will help

Faraz

We do need a lot more detail as already suggested so it is difficult to help. However, one other reason is that smeary bands could indicate protease activity: add protease cocktail.

Impurity in Ur sample could possibly be the reason