• Go to www.ncbi.nlm.nih.gov/blast/
• 
• Click on protein blast
• You will get a square box

• Give an accession number P01922 in this box,its an ID of human hemoglobin (when your sequence is already in database, you can give its ID).

• Select the swissprot protein sequences as a database.



• Select Psi-blast as an algorithm.

• Click on Algorithm parameter.



• Select 1000 for maximum target sequences.



• Select low complexity region for filters and maskin

• 

• Click on blast
• Search for leghemoglobin in first iteration (there will be no leghemoglobin in it).
• In first iteration it gives a NEW Symbol which shows you sequences that PSI-BLAST reports as hits for the first time.



• Click on go button for second iteration (there will be two leghemogloin and note down their E-value)



• Click on go for third iteration (there will be six leghemoglobin note down their E-value again, their E-value is lesser than the hemoglobin we obtained in second iteration).
• In third iteration it gives a GREEN PILLS which shows you sequences that PSI-BLAST has already used to obtain the current result.



• By iterating again and again bit score will increase and E-value will be decreased and we get better alignment between hemoglobin and leghemoglobin.

Contributed by:Aeman Zehra