Recently, I am performing Western Blot about Dopamine Receptor(DR), a GPCR (G-protein coupled receptors). And the band for DR in Western Blot was not so obvious as shown in Immunofluorescence.
It was said that GPCR as seven-transmembrane protein should be boiled as cytosolic proteins. And I often boiled the samples at 95 degree for 7 minutes.
After comparing the samples boiled or unboiled, Western Blot should significant difference. The boiled sample showed one-band while the unboiled samples showed two-band, nearby the band shown in boiled samples.
So, does anyone can give me some suggestions about GPCR or Dopamine Receptors study in Western Blot? Should I boil the samples or not?
Thanks a lot!
zhiyuan
My experience is with rhodopsin so I don't know if this is true for all GPCRs but if we heat a sample prep containing rhodopsin, especially in SDS, it will aggregate and a lot of it will not even enter the gel even though it is a 39K protein. Therefore, instead of heating, we used to let our sample prep in Laemmli buffer sit at room temperature for at least 30 minutes before loading. That means we also never froze samples in Laemmli buffer in the freezer either. We stored in another buffer and adde 5X or 2X Laemmli after thawing. I am pretty suer this is an issue with at least some other GPCRs.
Hi, as you have to do SDS-PAGE not Native-PAGE before western blotting you need to biol your samples at 95-100 C for 5-10 min. This will play role in denaturation of your proteins and also helps your samples to be mixed with dye. Therefore you have to boil your samples.
Is it a membrane fraction, or some lysate?membrane proteins tend to precipitate at higher temp. do you use some TX100 somewhere in the sampling process?
Hi Attila,
The sample is cell lysate. And I often use moderate lysis buffer "M-PER" from Pierce. I did not add extra TX100.
In that case, it is OK. Mightbe some more sample buffer needs or add extra SDS. A moderate heating is essential, but do not boil too long. The extra line at lower temp suggests some preserved structure.
The best way is to try different conditions in the first experiment: you can divide your sample into several tubes and incubate them at different temperatures for different time periods. You can try loading buffer with and without DTT, This is the protocol I used for D1R:
- detergent – 1% Triton X-100
- solubilization – rotate 1h, 4°C
- centrifugation: 14000 rpm, 30 min, 4°C
- take the supernatant (discard the pellet) – mix 1/1 with Leammli buffer 2x (+ 0.2 M DTT)
- 20 min, RT
- Load cca 50 mg of protein on gel
Good luck
I agree with Michaela's answer. I have performed relatively few Westerns myself on GPCRs, but my team regularly provided preparations of all kinds of membrane proteins to those that did for more than a decade. One of our most important conclusions was that there are no hard and fast rules on the optimum way to perform Westerns for such proteins. There are good starting points but it's always worth trying variations empirically, especially around temperature. This is not just fine tuning. Sometimes really low temperatures yield great results and boiling gives no bands at all, and vice versa. Zhiyuan, with issues on gels in general it helps if you indicate the apparent mwts of different bands, (or provide a picture). Extra bands can be caused by degradation, aggregation, or different recovery of glycosylated species. In this particular case testing different temperatures empirically may be the best first step regardless of the underlying issue. More detail would help to investigate in greater depth though.
My experience is with rhodopsin so I don't know if this is true for all GPCRs but if we heat a sample prep containing rhodopsin, especially in SDS, it will aggregate and a lot of it will not even enter the gel even though it is a 39K protein. Therefore, instead of heating, we used to let our sample prep in Laemmli buffer sit at room temperature for at least 30 minutes before loading. That means we also never froze samples in Laemmli buffer in the freezer either. We stored in another buffer and adde 5X or 2X Laemmli after thawing. I am pretty suer this is an issue with at least some other GPCRs.
I have found that for best results when doing WB using lysates there is no need to boil or even to heat the samples. This is true for membrane proteins and cytosolic proteins. We just mix lysates with equal volumes of 2X Laemmli buffer and use directly for WB. Try running boiled and non-boiled samples side by side.....
Thanks all of you, Michaela, Jim, Ellen and Kamil. You guys have provided me precious experience and suggestions. And I have tried to optimize the conditions with different boiling temperature or just at room teperature for 7 min and 20min.
zhiyuan
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