Problem with 2D gels

Harish Gangadharappa @Harish_Gangadharappa2

03 March 2013 5 9K Report

I am working on separation of mitochondrial proteins from human brain samples by 2D methods, but have not been able to get proper separation (discrete spots) on the gel.

Protocol followed

Separation of mitochondria by ultracentrifugation (6% Ficoll)Suspension of the pellet in PBS followed by Sonication~300ug of mitochondrial protein is precipitated by 30% TCA (Final)  pellet after centrifugation is washed with Ethylacetate:Ethanol(1:1) (twice) Resuspension in 200ul of Rehydratiion/Lysis buffer with DTT Centrifugation to remove any insoluble protein supernatant is loaded onto IEF tray with strip(11cm) for overnight active rehydration at 50v (16 hrs).

First dimension is carried out in Biorad IEF Protean cell; it generally takes ~18 to 20 hrs to complete.

Step 1: 250 V 30 min rapid ramp

Step 2: 8000 V 1 hr slow ramp

Step 3: 8000 V 40000 V-hr rapid ramp

Second dimension is carried out with 10% PAGE followed by Fixation and Silver staining

Masood Sepehrimanesh

Hi dear Harish

I worked on testes proteome and had similar problems but modified my protocol and obtained sharp spots:

* Active rehydration to passive (16 hrs and 30 mins)

* TCA precipitation to aceton precipitation (1:3 ratio)

* Also I used Tris-HCl buffer with sucrose instead of RH buffer

* The 1st dimension was run at seven steps and the voltage was increased step by step

* SDS-PAGE run at 12%T

Harish Gangadharappa

Thanks Masood Sepehrimanesh, could you please share the seven step 1st dimension protocol?

Masood Sepehrimanesh

Please follow these steps:

1- 0 to 50 V, Rapid, 15 min

2- 50 to 100 V, Rapid, 15 min

3- 100 to 150 V, Rapid, 15 min

4- 150 to 200 V, Rapid, 15 min

5- 200 to 4000 V, Linear, 2 hr

6- 4000 V, 36000 Vhr, Rapid

7- 500 V, 99 hr, for holding

After that, strips must be transfer to -70 degree of Celsius, overnight.

Sincerely

Harish Gangadharappa

Thank you Masood, that was really helpful

Parijat Sarkar

Hello Harish and Masood,

What was the pH range of your IPG strip?

I am using BIO-RAD pH 3-10 strips of 17 cm, in their manual they have mentioned one step protocol for first dimension i.e 40000-60000 Volt hours with end voltage 10000V. Do you think multiple steps have any advantages?

Thanks

Parijat

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