Hello everyone
I've recently been doing a study on a small molecular protein of human which is clustered near the membrane whose molecular weight is between 8-10 KD. I extracted the total proteins of several cancer cell lines with RIPA and detected them with WB in which 15% gel was used. But there was nothing on the PVDF membrane while GAPDH normally appeared. I have lentivirus which expresses a fusion protein whose molecular weight is between 35-45 KD but has same antigenic characteristics as my target protein and it also appears normally on the PVDF membrane with ECL reagent so I guess the primary and secondary antibody work well. But when it comes to target protein, no matter cell line or human tissue, there is nothing. I'm trying tricine-SDS-page method and I read a protocol (DOI: 10.1038/nprot.2006.4) which said Avoid boiling samples, because membrane proteins can irreversibly aggregate in SDS at temperatures >50 °C (bottom, 18 page). I never done this before cause I always mix the sample with SDS-PAGE protein Loading buffer 5X and boil it for 5-10 mins. So I have a question: What is the influence of irreversible aggregation in SDS for membrane protein? Why other proteins need boiling? Could SDS-PAGE protein Loading buffer be used without boiled?
And here is my SDS-page WB method:
1. Wash the cells with PBS for 3 times. Add 1 ml PBS to the cell culture bottle. Scrape the cells off the culture bottle on ice.800 rpm, 5 mins to collect the cells. 100 μL RIPA was used in which 1μL phosphatase inhibitor and 1 μL PMSF were added on the ice for 30 minutes. 13000 rpm, 20 mins and supernatant fluid is collected and mixed with SDS-PAGE protein loading buffer. Marker: 26616
2. Electrophoresis: 30 μg protein each tunnel. 80 V, 20 mins, then 120 V 40 mins. (The position of bromophenol blue is 6KD, so I cut the gel to get the gel between 6 to 20 before transfer. )
3. Transfer onto PVDF membranes: 90 V 15/30/60 mins (I tried three different time but none work. Could protein goes through the membrane if time is too long? There are people says 1 min for 1 KD to avoid the protein goes through. Does it true? And there are people told me they get clear result with 300 mA for 1.5h with the same protein, and they even do a knock down in the same cell line and get clear result which i got. Same gel and same antibody but I can't understand why they get it). PVDF membrane(0.2) which has been activated in methanol was used.
4. 5% skim milk prepared by TBST, 1 h. TBST, 10 mins, repeat three times.
5. Primary antibody, overnight. TBST, 5 mins, repeat three times.
6. Secondary antibody, 1 hour. TBST 5 mins, repeat three times.
7. ECL luminescence reagent. 0.1s to 2 mins.(and only GAPDH and fusion protein appear on membrane)
Here is my other questions which I hope you can help to resolve:
1. If my target works in transmembrane system but it's not stuck on membrane, does it belongs to membrane protein?
2. Could protein goes through the membrane if time is too long on 0.2 μm PVDF membrane? If so, how long should it take to avoid that situation?
3. What's the possible problem in my WB process which cause blank membrane? Is it degraded, or too little to be detected, or not extracted at all or diffused in the process? What should be done to verify and resolve this problem?
Hope you all have a good day. Thank you.
1. I am not a expert but from what I understand there are integral membrane proteins that are inserted and remain anchored to the memebrane via certain transmembrane topological features. Then there are peripheral membrane proteins that associate with the integral membrane proteins or the membrane directly. This can be by non-covalent protein-protein or protein-lipid interactions. You will need to scour the literature to see if other proteins with similar interactions like yours have been classified as peripheral membrane proteins.
2. Normally, the protein does not go through the membrane, but if the spot where the protein is being transferred gets saturated, then the excess protein may go through. If you are worried about losing protein, you can place a second membrane behind the first to catch the excess. Your 0.2 micrometer PVDF should be good enough to trap the 8-10kDa protein. They work well for Histones (11-15kDa). I use 45 min constant 200mA transfer for Histone blots.
3. How good are your antibodies against your target protein ? Is it a commercial antibody or is it polyclonal ? Since your GAPDH worked, the problem could be either with prescence of protein or quality of antibody. You can stain your blot with Amidoblack or Ponceau after transfer to check if the proteins are present at 8-10 kDa. If the antibody is good, then you can try incubation with another dilution at a higher titre.
I havent worked with membrane proteins but I am aware of the aggregation problems with integral membrane proteins. My suggestion would be to add loading dye to your sample and distribute them in PCR tubes. Set a series of gradient heating from 37oC to 60oC for 10 min or so. Hopefully your protein remains unaggregated in one of those temperatures. If your protein is relatievly good in one of those reactions you can vary incubation time while holding that temperature constant to find the best combination with minimum aggregation.
Hope this helps.
So regarding your questions with SDS, I work with membrane proteins and I do not boil the sample, it is true that sometimes you can have aggregations due to heating, due to the exposition of the hydrophobic zones. Try not to boil your sample.
Do you detect your protein in a Coomasie-stained gel or silver staining? This can allow you to determine wheather your protein is expressed and extracted properly.
Sometimes proteins can pass through, but since your GADPH is there, it may mean that the transfert has been properly done. I transfer at 200 mA for 75-90 mins (nitrocellulose membrane).
So check whether your protein is detectable on gel while not boiling the sample, try staining your membrane with Ponceau red.
If your protein is of low-abundance, try to enrich it : since you think it is a membrane protein, centrifuge for 1h at 100.000 xg, and the pellet will contain membrane proteins : resuspend them and analyse that by SDS to see if it changes something or not.
I agree with Jorgaq Pata for the ponceau staining of the membrane right after blotting, this will prove if you even have proteins on the membrane or not. Also in our lab for secondary antibody we use all-phosphatase antibodies and develop the bands directly on the membrane, and in this technique even the faintest bands are visible (and you can leave it to develop overnight).
also as you have small protein, try to use membranes with smaller pores for blotting, this might also help.
Thank you very much for every suggestion.I solved this problem a few days ago.Here is my suggestion for who has similar problems.
1.Use 10% SDS-PAGE system, DO NOT USE 12%,15% OR TRICINE-SDS-PAGE, cause this will diffuse your protein. In fact, small molecular proteins do not completely follow the motion law of the bigger molecular proteins. Unless you soak a large part of your membrane in your primary antibody, you are likely to lose it.
2. Shorten electrophoresis time. My experience is for 10% SDS-PAGE system, 80 V 30mins, then 120V 50min.
3. Change your antibody after try different concentration. I used antibody from SANTA CRUZ at first, it works at the concentration of 1:200 which does not mean that there is a problem with its quality, but the concentration is still too high for WB. After used once, it was invalid. Then I bought another antibody.
4.BOIL OR NOT BOIL. I tried both and they both works in WB.
Thank you again for your suggestions.
@Niloufar Pirayesh @Praveesh Valissery @Jorgaq Pata
Dear Ma Yl
I also have the same problem.
I dont understand why you use the 10% gel instead of 12%, or 15%? I think the higher the percentage, it will separate the protein well. What do you mean by the protein will diffuse?
For answer number 3, what do you mean by the concentration is still too high?
My protein is 17.5kD. I already found it but still blurry. Do you have any suggestions?
thank you in advance
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