I have performed a SDS-PAGE using wasp venom and most of the proteins remained on the top of the well without being gone through the gel. Is there any specific reason for this aggregation? and how could I overcome this problem?
You have to more specific to get a good answer. How did you prepare the samples? How did you run the gel. Aggregation could be due to a number of things. How much protein you load in the gel and what is the size of the protein of interest? You need to provide more info.
you may need to check the composition of your protein extraction buffer and composition of SDS & β-mercaptoethanol also.
If your protein is not temperature sensitive, you can boil the protein after adding extraction buffer at 95°C for 10 mins and load it on the gel (SDS-PAGE).
I share the suggestions given above. In addition, what was the concentration of the gel in percentages? 10%? 12%? 14%? What about the molecular size of your target protein in kDa? If the molecular size of your target protein is high and the concentration of your gel is also high, your protein band will be concentrated at the top. What about the quality of your sample buffer? Does it contain the appropriate concentration of protein denaturing agents, such as SDS, DTT etc? What about the purity of your protein? Did you load the cell lysate? or purified protein? Do you have idea about the physicochemical properties of your target protein, such as hydrophobicity, surface charge etc?
I agree with some of the suggestion given by Himesh Makala, Alemu Tekewe for running gel. In addition I want stress that you have to check for the DNA in your protein sample. your protein sample will be viscous if you have DNA in it and you will have trouble in proper loading. There is simple way to remove DNA from protein sample, Just pass your sample through 22-25G needle four to five times and then centrifuge 10000g for 30 min. collect sup which will be DNA free protein.
Aggregation/precipitation can occur for a number of reasons so it is diffcult to say for sure why.
however:
1. heat sample at 65C for 15 minutes instead of 95C. We have found that higher temperatures for some proteins causes degradation/aggregation. spin sample to pellet insoluble debris.
2. Increase SDS concentration. You need to make sure protein is saturated in negative charge. Especially true if you have a high protein concentration.
protein you extract might be not pure. your sample preparation seems to be the problem. solve the peoblem step by step starting from sample preparation. can you upload the image you got after running samples. it will help everyone to analyse and giving appropriate and specific suggestions.
Thank you all for your suggestions, The samples were wasp venom proteins which were collected by electrical stimulation of the insects. So the samples do not containing other contaminants other than the insect venom. I put 25 micro grams of venom to a well. Sample buffer contained β-mercaptoethanol to denature the venom proteins. I am looking up a range of proteins in the venom sample about 25 KDa to 100 KDa in size. I used 12% gel with 5% stacking gel run the gel at 70 V for about 2 h. when mixed the sample with sample buffer, protein clots were appeared and did not removed even the samples were heated to 95c for 5 minutes and I tried 65C for 15 min, as suggested, how ever, the clots did not removed. I think the formation of protein clots would be the problem. Do you all have any suggestion for removing the protein clots?
Have you tried any of the above suggestions? Any success? Have you seen any protocols for sample preparation and electrophoresis in the literature. Try to duplicate the most recent one. "Stand on the shoulders of giants"
Try put you sample in caotropic agents like urea 8M or DTT, or both, before SDS-PAGE. Try put enough 2-ME at loudding buffer and 10 minutes of boiling.
IIRC, the poisons in bees and wasps contain proteins with high positive charge. You may get more luck with electrophoresis in positive detergents, e.g.
@ARTICLE{Bux-03,
title = {Cationic Electrophoresis and Electrotransfer of Membrane Glycoproteins},