Repeated flash freeze and thaw
In order for the protein to enter the separation gel, the sample must be lysed, so the first stage of sample preparation is lysis
.
Proteins expressed within cells are extracted by lysis, which destroys the plasma membrane and/or cell wall of the cell to release the protein
.
Extracellularly expressed (secreted) proteins do not need to be lysed, although cells can be processed to observe proteins
captured due to improper synthesis.
Large-scale protein purification – Extraction methods
Proteins from various sources can be analyzed by Western blotting
.
Samples may come from protein expression systems, such as mammalian or bacterial cell cultures, or from clinical tissue samples, each requiring a different treatment to produce a usable imprint
.
The method used for protein extraction depends on the nature of the
sample.
Protein expression occurs in different types of cells such as mammals, insects, bacteria, and yeast, and some samples may come from tissues; These samples are structurally specific and therefore require different treatments
.
Some proteins
in mammalian or insect cell systems may be expressed in the supernatant and therefore do not need to be extracted
from the cells.
tip: Keep the supernatant sample and load it onto the gel next to the cell lysate when screening expression
.
Screening and processing
protein expression is usually screened
by western blotting.
For example, the representations of several clones can be compared to each other, or they have different expressions in different expression
systems.
In these screening tests, samples are removed from protein samples and loaded for analysis
.
The supernatant is usually compared to the cell pellet to locate where
the expression occurs.
The total protein observed in Coomassie staining can be compared
with the expression of target proteins specifically detected in Western blots.
The basic treatment performed in a screening test may not represent how the final expression is processed for final use, such as crystallography tests that require finer purification steps
.
Figure 1: Detection of His-tagged proteins in a series of cell lysates
.
Add the C-terminal His-Tagged protein to the cell lysate at
a concentration of 100 ng.
Cell lysates of HEK 293, CHO, BL21 E.
Coli and Sf9 insect cells are reduced and boiled at 100 °C for 5 min before loading into tandem SDS gel at
9 μg/well.
A.
Western blotting
.
The gel was electroblotted onto a nitrocellulose membrane, blocked with 5% BSA in PBS/0.
2% Tween 20 and probed with Jackson ImmunoResearch's HRP Rabbit Anti-His Tag (300-035-240) diluted at 1:20K and visualized
using a digital imager.
B.
The gel is stained
with Coomassie.
The purpose of lysis buffer
is to destroy cell membranes to release proteins
.
Lysis buffer typically consists of a detergent that destroys the lipid bilayer
of the cell membrane and forms a micelle.
There are many buffer formulations that have been validated for different cell types or protein expression locations, such as lysate RIPA or NP-40
.
The processing of the material should be carried out on ice to minimize protein degradation and denaturation
due to cellular components.
Proteases and methods of inhibition
of lysis, such as those involved in the mechanical destruction of cells, lead to the release
of proteases within cells.
These proteases can digest and truncate proteins of interest, which can result in multiple bands
observed on the membrane.
The sensitivity of proteins to proteases depends on their amino acid composition, and intracellular proteins are less
resistant than proteins on the surface or outside of cells.
When membrane proteins are dissolved by detergents, they are particularly susceptible to degradation
by proteases.
Bioinformatics tools can predict in advance the sensitivity of proteins to protein hydrolysis
.
Protease inhibitors can be used to prevent proteolysis; They can be a mixture of chemical inhibitors and enzyme inhibitors that can be added to the lysis buffer before being added to the cells or buffers in which the proteins are stored
.
There are a range of inhibitors that can work
on common species of serine, cysteine, aspartate protease, as well as minolpeptidase and metalloproteinase.
Different expression systems or sensitivities to inhibition of protein activity may prevent the use of certain inhibitors
.
Proprietary mixtures of protease inhibitors can usually be used, or separate reagents may also be applied
.
In buffer selection, azide can also be a source of proteases to prevent bacterial growth
.
Dephosphorylation may occur after
the cells have been lysed.
If the phosphorylated protein is the target protein for western blotting, then phosphatase inhibitors, such as sodium vanadate, should also be added to the lysis buffer
.
Samples should be refrigerated throughout sample preparation to minimize the possibility
of degradation and dephosphorylation.
The following table lists some examples of inhibitors and additives:
| depressor |
protease |
Precautions |
EDTA
(Ethylenediaminetetraacetic acid) |
Metalloproteases |
Incompatible with some affinity chromatography columns, it is possible to inhibit enzymes that require divalent cations |
| Pepstatin |
acid proteases–pepsin |
|
| Leupeptin |
Serine and Cysteine proteases |
Toxic |
TLCK
(nα-tosyl-l-lysine chloromethyl ketone hydrochloride) |
Serine proteases (Trypsin like proteases) |
Toxic/unpleasant odor |
PMSF
(Phenylmethylsulfonyl fluoride), AEBSF is a common water-soluble version with more stable properties |
Serine proteases |
Neurotoxins
.
Short shelf life and comes with.
|
| Azide |
Bacterial growth |
Interferes with downstream processing/inspection
.
Toxic
.
|
| Sodium orthovanadate |
ATPase, alkaline phosphatase and tyrosine phosphatases |
Toxic |
| Sodium pyrophosphate |
Serine/threonine phosphatases.
|
Corrosive and irritating |
The pH of the pH
lysis buffer is important for control during sample preparation to ensure stability and protein activity is maintained
.
By using buffers with the correct pH, protein stability and solubility are ensured, preventing aggregation and precipitation
.
Related to stability is the activity
of the protein.
Using the correct pH ensures that the protein is left for downstream use
.
The pH of the buffer is usually one pH higher or lower than the protein isoelectric point, usually in the physiological range
between pHs 6 and 8.
Use a combination containing a weak acid with its conjugated base, or a combination
of a weak base and its conjugated acid.
The table below lists common buffers and their pH ranges
.
| Buffer |
pH range |
| Citric acid – NaOH |
2.
2 – 6.
5 |
| Sodium citrate – citric acid |
3.
0 – 6.
2 |
| Sodium acetate – acetic acid |
3.
6 – 5.
6 |
| Cacodylic acid sodium salt – HCl |
5.
0 – 7.
4 |
| MES – NaOH (2-(N-morpholino)ethanesulfonic acid) |
5.
6 – 6.
8 |
| Sodium dihydrogen phosphate – disodium hydrogen phosphate |
5.
8 – 8.
0 |
| Imidazole HCl |
6.
2 – 7.
8 |
| MOPS – KOH (3-(N-morpholino)propanesulfonic acid) |
6.
6 – 7.
8 |
| Triethanolamine hydrochloride – NaOH |
6.
8 – 8.
8 |
| Tris – HCl ( Tris(hydroxymethyl)aminomethane) |
7.
0 – 9.
0 |
| HEPES – NaOH(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) |
7.
2 – 8.
2 |
| Tricine – NaOH |
7.
6 – 8.
6 |
| Sodium tetraborate – boric acid |
7.
6 – 9.
2 |
| Bicine – NaOH |
7.
7 – 8.
9 |
| Glycine – NaOH |
8.
6 – 10.
6 |
Tween reagents
Nonionic detergents can be used to increase the solubility of non-polar, insoluble proteins
.
Examples include Triton™X-100 and Tween(r)-20
.
Osmolality stabilizers When purifying proteins from specific subcellular structures, such as organelles from eukaryotic cells, lipid membranes, or proteins
from the bacterial periplasmic space, osmotic stabilizers, such as high concentrations of sucrose
, need to be added to the cell lysis step.
Their addition helps stabilize subcellular structures during lysis and also prevents cell lysis
during cell wall degradation.
Centrifugation is performed under specific gravity of the isolated subcellular structure and density gradients (such as sucrose or glycerol fractionation) are added, usually further enriching the proteins
in the subcellular structure at western blotting.
The presence of an osmotic stabilizer may affect how proteins migrate on the gel, and samples may need to be diluted into buffers of different osmotic strengths prior to loading
.
Salts Salts are
added to vary the ionic strength
of the buffer solution.
Adding salt can improve protein solubility; However, too much salt can reduce solubility and precipitate proteins
.
By optimizing the ionic strength of the solution, proteins can be encouraged to maintain their folded conformation, thus preventing damage
caused by proteolysis by preventing exposure of vulnerable internal residues.
Stabilization of the surface charge prevents protein aggregation
.
Sample Clarification
Use centrifugation to clarify the sample
.
It can be used after lysis, thereby overcentiving the cell lysate at a very high rate for a long time to pellet cell debris and chromosomal DNA, which is then discarded and retained in the solution
containing the dissolved protein.
For proteins that reach the medium on the surface of cells, such as in mammalian cells, the speed and duration of centrifugation must be adjusted to provide less destructive conditions to prevent cell lysis
.
Centrifugation, usually through density gradients, is also used to isolate cell parts
.
For example, microsomes (endoplasmic reticulum, plasma membrane) can be precipitated
at high speed by low-speed centrifugation after initial clarification.
Improperly clarified lysates and cellular component residues with high NaCl concentrations can lead to incorrect protein separation in the sample during SDS-PAGE, resulting in blurred bands or egg acquisition of proteins
of non-molecular weight of interest.
The presence of nucleic acids can also clog pores and affect sample migration, so deoxyribonuclease (DNase) can be added to eliminate this contaminant
.
Dialysis or gel filtration can also be performed to reduce NaCl concentration
.
These purification techniques enable more accurate separation during electrophoresis
Sample Concentration
Ideally, the total protein concentration
of a sample should be determined using methods such as Bradford, Lowry, or dicinoxonic acid (BCA) assay prior to loading.
The predetermined sample concentration enables the wells in the separating gel to be loaded with the same volume and concentration to optimize consistency
across the gel.
If too many proteins are loaded into the wells, the migration of proteins in adjacent lanes can be affected, making it difficult to interpret the results
.
Depending on the size of the wells, a total protein amount of 10-50 μg per lane is sufficient
.
However, if pure protein is used, this weight may be too concentrated
.
Low-abundance proteins may need to be concentrated to facilitate western blot detection
.
Purification labeling (such as HIS6 labeling) allows protein
concentration using affinity columns such as nickel.
It is important to ensure that the elution buffer is
compatible with the loading buffer.
In addition, if there are antibodies that target specific proteins, methods such as immunoprecipitation can be used to efficiently concentrate the proteins
.
Protein concentration
Large protein preparation often requires a concentration step in a purification step to reduce the material volume to a more manageable size
.
Affinity, ion exchange, and metal chromatography or tangential flow filtration can be used to remove excess liquid volumes and concentrate total or proteins of interest
.
WB samples may not require a concentration step because the abundance of the protein is unlikely to fall below detection limits
due to the sensitivity of the technique.
Optimal Protein Concentration
Loading too much protein into the wells can lead to accidental protein migration, but improper settings can also lead to protein migration, such as when the concentration of salts or denaturants (such as guanidine hydrochloride) in the loading buffer is too high
.
In addition, gel operating conditions that raise temperatures excessively during gel or Western blot runs (e.
g.
, voltage or salt concentrations in the running buffer are too high) can negatively affect
protein separation.
See Chapter 4 to learn how to resolve related technical issues
.
The following table lists some common sample preparation issues
| Effect |
Cause |
Solution |
| Gummy or viscous sample/ poor gel entry or migration |
DNA |
DNAse |
| Loading dye changing color |
pH |
Buffer exchange or addition of acid/base |
| Diffuse band |
Low or high salt |
Buffer exchange |
Loading conditions and buffers
Before loading, mix the sample with the loading buffer, which helps to denature the protein and load the sample into the separation gel
.
It is usually a combination
of dyes, reducing and denaturing agents, and glycerol.
The samples are then boiled at 100 °C for 5 min and then briefly centrifuged
before loading.
In some cases, for highly hydrophobic integrin proteins, a temperature below 100 °C is required
.
Stain Loading
Adds loading dye to the sample to visualize loading and sample migration
during electrophoresis.
A commonly used loading buffer, Laemmli loading buffer, is a combination
of bromophenol blue, glycerol, Tris, SDS, and reducing agents such as DTT or BME.
Due to its small size, bromophenol blue migrates faster than proteins in the sample and provides a migration frontier to monitor the electrophoresis process and prevent sample loss
.
It can also turn yellow if acidic conditions are encountered, indicating inadequate buffering systems or omitting the treatment step
.
Glycerol makes the sample denser than the running buffer, allowing the sample to "sink" to the bottom of the
well.
After boiling the sample in loading buffer, centrifuge
the sample briefly.
V.
Oxidation and Reduction Conditions
Samples analyzed by WB are denatured and reduced so that proteins are decomposed
according to their molecular weight during electrophoresis.
The addition of reducing agents, such as β-mercaptoethanol (BME) or dithiothreitol (DTT), reduces disulfide bonds between cysteine residues
.
SDS treatment denatures proteins by breaking non-covalent bonds within and between residues, thereby linearizing proteins into "fluffy" chains
of amino acids.
SDS also effectively saturates the polyamide backbone of proteins, reducing the effect
of charge on protein migration.
Precautions
Incomplete reduction of the sample may cause the protein to fail to resolve as expected, which may be observed as a trailing band or a band
of undesirable size.
Therefore, fresh reducing agent should be added to the loading buffer on the day of
use.
Insufficient heating can also lead to incomplete denaturation of the
sample.
Natural or non-reducing gels
Non-reducing or natural gels are used to observe the natural behavior of proteins, such as their stoichiometry or interaction with other proteins
in solution.
These proteins are usually observed
by Coomassie or silver staining rather than western blotting.
In this case, the reducing agent should not be added to the loading buffer, SDS removed from the loading and running buffer, and the sample
should not be boiled.
Table 5 details the components
in the buffer according to the desired protein state.
Bromophenol blue is also often added to the loading buffer to visualize
protein migration during gel electrophoresis.
Due to its small size, bromophenol blue migrates faster
than proteins in the sample.
The following table compares the protein and buffer compositions in different states:
| Protein State |
Sample Loading Buffer |
Gel Running Buffer |
| Reduced and denatured |
SDS βME or DTT
Boil 5–10 minutes*
*for integral membrane proteins, lower temperature (e.
g.
70 degrees C) may be preferred |
SDS |
| Reduced and native |
βME or DTT |
No SDS |
| Oxidized and denatured |
SDS Boil 5–10 minutes |
SDS |
| Oxidized and native |
|
No SDS |
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