protein resuspension buffer

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amolgaikwad
amolgaikwad's picture
protein resuspension buffer

Hi there,

I'm working with rice proteins, n after isolation of crude protein from rice leaf tissue samples by TCA method, I get the protein white powder.. but I'm not getting the appropriate resuspension buffer to resuspend the proteins as I'm going to do western blotting of that.......

Can anybody help me for this???????????????????????

protoldo
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Hola, I never have dry TCA

Hola, I never have dry TCA pellet, and I don´t know if you have neutralized it before dry. The TCA pptation to do PAGE, produce a pellet dificult to resuspend, but imposible to disolve. tMy method, suspend the pellet in adequate volume of water pippetig better than vortexing , add loading buffer, if you haven´t neutralize before the sample turns yellow. Add Tris base  or tris pH8.8 (1-2M) untill the sample turns blue again. to 10ul of aquous suspension, 10ul of sample buffer and 3-5 ul of neutralizing tris solution. Good luck

amolgaikwad
amolgaikwad's picture
Thanks protoldo for your kind

Thanks protoldo for your kind reply,

I got your idea to resuspend the protein solution n I wil definitely try that......

Actually I used TCA(trichloroacetic acid)(10% w/v)+0.07%v/v 2-mercaptoethanol in chilled Acetone for precipitating the proteins(incubation at -20C for 2 hrs) n then gave washes by cold acetone having 0.07% 2 ME to remove pigments n lipids until pellet become colourless.....

But actually I dont know the role  n pH of TCA here. (made 100% w/v TCA by dissolving 10gm TCA in 5.5ml milliQ water).

So can you please explain the role of TCA over here?????

Once again thanks for the reply and the suggestion,,,,

protoldo
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The main reason is that all

The main reason is that all proteins precipite in acid medium, I don´t know the specific reason for the use of cold TCA, but it´s used from years. One more thing in order to avoid accidents, TCA hasn´t to be wheight. if you have a new 500g jar, you have to add water to the original container untill 500ml, and you have a 100% solution without manipulate it. Good luck

amolgaikwad
amolgaikwad's picture
hey protoldo

hey protoldo

I used ur method n got good results, but the main problem is that the quantity of protein i got by TCA method is very less-- 0.6mg/ml n the protein of my interest is expressing at low levels(i herd that there is half amount of rubisco in crude extract here.) n as my antibodies against protein of ineterest is just the part of serum, not that much pure in form.......

I'm getting difficult to detect my protein through western blotting(i detected +ve control till 0.3 micro gram).

so is there any method/protocol for enough protein isolation or to concentrate the protein sample? (is dyalysis used for concentrating proteins?)

wting for reply....

protoldo
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Hola, the first idea to

Hola, the first idea to increase the amount of precipitate is increase the amount of starting sample. two, the sample could be concentrated by filter devices with controlled pore membrane; there are from 3Kd. to 100Kd and there are devices for small volumes with centrifugation to high volumes by gas pressure untill tangential ultrafiltration that isn´t your case. See the Millipore-amicon page. Dialisys tube isn´t the most adequate method, but you can use it. Knowing that the pore size of the normal tube is about 12 Kd, you can fill the bag and put it in an absorbent medium like dextran or paper towels  waiting that enough liquid goes out the bag, increasing the protein concentration inside, this is valid if your protein has a MW higher than 20 KD because the pore size isn´t exact and the protein conformation has a role in the pass across the bag. Good luck

amolgaikwad
amolgaikwad's picture
hey hi,

hey hi,

As i have  much sample, i will start with new sample. But the problem is there is low expression in leaf compared to root, so can i use this TCA protocol for  root protein isolation? (But there are no pigments in root as i used acetone wash to remove those pigments n lipid contents...)

protoldo
protoldo's picture
Yes if you need concentrate

Yes if you need concentrate the sample you can precipitate it with TCA. I would try precipitation of the first homogenate without any extraction. Good luck