Is the construct designed for secretion into the culture medium, or is this intracellular expression? If the latter, beware that it is very difficult to unambiguously spot a recombinant protein in SDS-PAGE of crude yeast lysates. Try to express at least part of the protein in E. coli (or have a peptide conjugated to a suitable carrier) in order to raise a suitable antiserum for detection. You will save time in the long run.

Dear Martin,

Thanks for the reply and sorry for my late response.  

The construct is made for the intracellular expression. I completely agree with you. It is very difficult to spot recombinant protein in SDS-PAGE. I dont have an option to go for a western blot due to the antibody issue. Will try to express in Ecoli as you said.

Sajeevan

Here's a protocol we routinely use for probing yeast extracts.

1. Resuspend pellet from 5ml BMGY/1ml BMMY culture in 400 ul water

2. Transfer 100 ul to Eppendorf tube

3. Add 100 ul 2 mM NaOH

4. Incubate 5 min at RT

5. Pellet (3 min, max speed benchtop microcentrifuge)

6. Make up 1x Laemmli Buffer from 4x stock with water and add reducing reagent

7. Remove all NaOH from tubes using a pipette

8. Resuspend pellet in 50ul Laemmli Buffer

9. Boil for 3 min at 95C

10. Pellet again

11. Load 15 ul of the supernatant onto gel

Hope that's helpful

HI Sayeevan,

there are several commercial buffers that you can use, i used the bugbuster protein extraction reagent for protein expressed in bacteria, but there is a buffer for yeast it works very good and the extraction level is high

below the link

71186 | YeastBuster™ Protein Extraction Reagent (Efficient extraction of protein from yeast without mechanical disruption and enzymatic lysis) Merck

Best regards

Bogdan

Thanks Darren Leneghan and Bogdan Munteanu for the suggestions.