9 replies to this topic

[alicee!]

hi all..

the standard protocols for sdspage involve addition of sample buffer to the protein sample and then boiling the samples for 2 mins.. before loading on to the gel.
is there some specific reason for boiling the samples.
is this specific for reducing samples? or is applicable to both nonreducing and reducing conditions.

why and for how long should the samples be boiled. waht decides the time?

thanks..

[Dominic]

gets rid of the snot

(try not boiling - you'll see what i mean)

dom

[K.B.]

High temperature denatures protein and promotes denaturation by SDS, it also breaks disulfide bonds and promotes (accelerates) reaction with reducing agenst (eg. DTT, bME).

[alicee!]

K.B., on Jul 31 2009, 07:53 AM, said:

High temperature denatures protein and promotes denaturation by SDS, it also breaks disulfide bonds and promotes (accelerates) reaction with reducing agenst (eg. DTT, bME).

i tried no boiling n boiling for different times.. 2 min 5 min n so on..
when i donot boil my samples i donot see a low molecular weight band.. and when i boil.. it increases with increase in time for boiling.

also, higher the amount of sds in the sample buffer.. lesser the low molecular weight band intensity is..

any ideas.. ?
how do i know whether this band truely exists or not?

[madrius1]

Boiling for too long may result in protein degradation. This may explain the band.

As for the sds.. I don't know!

[cellcounter]

alicee!, on Aug 2 2009, 10:51 PM, said:

K.B., on Jul 31 2009, 07:53 AM, said:

High temperature denatures protein and promotes denaturation by SDS, it also breaks disulfide bonds and promotes (accelerates) reaction with reducing agenst (eg. DTT, bME).

i tried no boiling n boiling for different times.. 2 min 5 min n so on..
when i donot boil my samples i donot see a low molecular weight band.. and when i boil.. it increases with increase in time for boiling.

also, higher the amount of sds in the sample buffer.. lesser the low molecular weight band intensity is..

any ideas.. ?
how do i know whether this band truely exists or not?

1. This is a matter of optimization (i.e. you try out many conditions - no boiling, some boiling, more boiling, less SDS, more SDS etc). Some proteins, depending upon their physical properties and chemical make-up, work best with boiling, some don't (especially the membrane proteins).

2. As a rule of thumb (not always applicable), the higher the MW, the more boiling time, more SDS concentration is required.

How do you know?

1. If it is a specific product and a specific band coming up with only your specific antibody and not with IgG,

2. If it is the expected size,

3. If it vanishes with well-controlled competetion experiments,

4. and lastly, if nothing else is sure, if you sequence the protein,

you know it is correct.

Edited by cellcounter, 04 August 2009 - 07:20 AM.

[alicee!]

cellcounter, on Aug 4 2009, 07:18 AM, said:

alicee!, on Aug 2 2009, 10:51 PM, said:

K.B., on Jul 31 2009, 07:53 AM, said:

High temperature denatures protein and promotes denaturation by SDS, it also breaks disulfide bonds and promotes (accelerates) reaction with reducing agenst (eg. DTT, bME).

i tried no boiling n boiling for different times.. 2 min 5 min n so on..
when i donot boil my samples i donot see a low molecular weight band.. and when i boil.. it increases with increase in time for boiling.

also, higher the amount of sds in the sample buffer.. lesser the low molecular weight band intensity is..

any ideas.. ?
how do i know whether this band truely exists or not?

1. This is a matter of optimization (i.e. you try out many conditions - no boiling, some boiling, more boiling, less SDS, more SDS etc). Some proteins, depending upon their physical properties and chemical make-up, work best with boiling, some don't (especially the membrane proteins).
yes but different conditions do show different results.. it is difficult to say which one of these is a true result.
meaning.. no boiling.. vs. boiling 2 mins. 5 mins.. n up to 10 mins.. the lower molecular band increases in intensitiy. it is difficult to say if this is already present in the sample (some how bound/associated with the principal protein) and is dissociated in presence of the sample buffer and the boiling treatment.
same is with the sds.. higher sds to protein ratio.. lower the low MW band intensity. it is difficult to say whihc is the true result.
it would be inappropriate to not report the impurity if it is present. also the true quantity of the impurity needs to be known but the procedural problems are not answering these questions.

2. As a rule of thumb (not always applicable), the higher the MW, the more boiling time, more SDS concentration is required.
mine is near 19 kDa. I see an extra band near 14-15 kda.

How do you know?

1. If it is a specific product and a specific band coming up with only your specific antibody and not with IgG,
Yes it is product specific, confirmed with an immunodetection on a western blot.

2. If it is the expected size,

3. If it vanishes with well-controlled competetion experiments,

4. and lastly, if nothing else is sure, if you sequence the protein,

size of the protein: 19kda
low Mw band obtained: 14 kDa.
mostly with intact N terminal sequence. may be truncated at C terminal. Am unable to sequence this.
Not able to answer if this a true band or generated due to sample treatment.
If I knew the site of cleavage, how would that help confirm how much of it is present.

you know it is correct.

thanks.

[THE ROCK]

Any protein sample in case of reducing SDS PAGE, sample has to boiled with SSB for atleast 5 min.The boiling will enhance the action of SDS,therefore the tertiary structure of proteins becomes primary and break in disulfide bond by reducing agent in SSB will lead form single band for separation based on charge and molecular weight in gel.

[Bill Nye]

How about spinning down after the boil, how long do you spin for and at what temperature and RPM? Are the benefits to spinning longer?

[Nrelo]

Bill Nye, on Sep 1 2009, 04:19 PM, said:

How about spinning down after the boil, how long do you spin for and at what temperature and RPM? Are the benefits to spinning longer?

just to spin down any ppt formed during sample preparation. I usually spin at full speed (13,000rpm) for 5min and load the supernatant.